Prevention and Treatment of Mycobacterium Infection

ABSTRACT

The invention relates to the identification of antigens, including  Mycobacterium  sulphate assimilation pathway components such as CysD, for preventing and treating  Mycobacterium  infection, especially but not exclusively  Mycobacterium tuberculosis  infection; to expression systems including live  Mycobacterium  for expression of said antigens for prevention and treatment of said infection; and to use of said antigens and expression systems for prevention and treatment of said infection.

FIELD OF THE INVENTION

The invention relates to the identification of antigens for preventingand treating Mycobacterium infection, especially but not exclusivelyMycobacterium tuberculosis infection, to expression systems includinglive Mycobacterium for expression of said antigens for prevention andtreatment of said infection, and to use of said antigens and expressionsystems for prevention and treatment of said infection.

BACKGROUND OF THE INVENTION

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB)claims almost 2 million lives annually ((Dye, 2010) and globally is theleading cause of death due to a single bacterial agent. The situationhas become more critical in the past decade due to co-infection with HIVand the inefficiency of the current vaccine, Bacillus Calmette-Guérin(BCG), to protect adults against TB ((Dye, 2010).

The identification of novel and protective antigens recognised byinfected individuals would represent a major advance in the control ofTB and may form the basis of new therapeutics to limit disease spread.

Current TB vaccines in clinical trials include viral-vectored oradjuvant-based subunit vaccines, as well as whole mycobacterialvaccines, that express one or more immunogenic M. tuberculosis antigens((Kaufmann, 2011). Most of these strategies are based on secretedantigens of M. tuberculosis that are presumably more readily ‘seen’ bythe host immune response (reviewed in ((Kaufmann, 2011)). The protectiveefficacy in humans of these new candidate vaccines yet to be determined.

There remains a need for further candidate antigens and vaccines forprevention and/or treatment of Mycobacterium infection, especially M.tuberculosis infection.

Further to identification of antigens, the targeted modulation ofantigen expression in antigen presenting cells by recombinant vaccinevehicles such as Bacille Calmette Guerin (BCG) would significantly aiddevelopment of effective immunotherapeutic strategies.

There remains a need for candidate expression systems, especially thosecapable of an immediate and sustained expression of protective antigen,thereby enabling improved antigen specific immune responses toMycobacterium, especially M. tuberculosis.

SUMMARY OF THE INVENTION

The invention seeks to address one or more of the above mentioned needsand/or provide improvements in the prevention and/or treatment ofMycobacterium infection and in one embodiment provides a method forminimising the likelihood of development of a Mycobacterium infection inan individual including:

-   -   forming an immune response to a component of a Mycobacterium        sulphate assimilation pathway (SAP) in an individual;

thereby minimising the likelihood of development of a Mycobacteriuminfection in the individual.

In another embodiment there is provided a method for providing anindividual with immunity to Mycobacterium infection including:

-   -   forming an immune response to a component of a Mycobacterium SAP        in an individual;

thereby providing the individual with immunity to Mycobacteriuminfection.

In another embodiment there is provided a method for preventing aMycobacterium infection from developing in an individual including:

-   -   providing a component of a Mycobacterium SAP in an individual        requiring said prevention in conditions for enabling formation        of an immune response to said component in said individual;

thereby preventing the infection from developing in the individual.

In another embodiment there is provided a method for treating anindividual having a Mycobacterium infection to at least minimise theprogression of the infection or a condition associated with theinfection including:

-   -   providing a component of a Mycobacterium SAP in an individual        requiring said treatment in conditions for enabling formation of        an immune response to said component in said individual;

thereby treating the individual.

In another embodiment there is provided a use of a component of aMycobacterium SAP in the manufacture of a medicament for minimising thelikelihood of development of a Mycobacterium infection in an individual.

In another embodiment there is provided a use of a component of aMycobacterium SAP for minimising the likelihood of development of aMycobacterium infection in an individual.

In another embodiment there is provided a method for determining whetheran individual is immune to a Mycobacterium including:

-   -   providing a component of a Mycobacterium SAP in an individual in        conditions for enabling formation of an immune response to said        component in said individual;    -   determining whether the individual develops a protective immune        response to said component;

wherein development of a protective immune response determines that theindividual is immune to a Mycobacterium;

thereby determining whether the individual is immune to a Mycobacterium.

The Mycobacterium CsyD gene or gene product is a preferred component foruse in the above described methods.

In another embodiment there is provided a vaccine or immune stimulatingcomposition for providing an immune response to Mycobacterium in anindividual including:

-   -   a component of a Mycobacterium SAP;    -   a compound for potentiating an immune response to the component        of the Mycobacterium SAP.

In another embodiment there is provided a vaccine or immune stimulatingcomposition for providing an immune response to Mycobacterium in anindividual including:

-   -   a Mycobacterium cell including a recombinant CysD gene or        protein.

In another embodiment there is provided a recombinant protein suitablefor use in the above described composition, said protein including afirst region having a sequence encoded by a Mycobacterium CysD gene andone or more further regions having a sequence of a Mycobacteriumantigen. Also provided is a nucleic acid encoding the recombinantprotein, and an expression vector including said nucleic acid.

In another embodiment there is provided a protein suitable for use inthe above described composition including a first region having asequence encoded by a Mycobacterium CysD gene and a further regionhaving a sequence encoded by a Mycobacterium Agb85 gene. Also providedis a nucleic acid encoding the protein and an expression vectorincluding said nucleic acid.

In another embodiment there is provided an expression vector including anucleic acid encoding a Mycobacterium CysD protein and a promoter,wherein said promoter is operably linked to the nucleic acid forexpression of the nucleic acid when a Mycobacterium strain including thevector is introduced into an APC, said promoter having a sequence of aMycobacterium promoter that causes expression of a Mycobacterium ATPindependent chaperone.

In another embodiment there is provided a Mycobacterium including arecombinant protein, nucleic acid or expression vector described above.The cell may be derived from M. tuberculosis or may be an attenuatedstrain of M. tuberculosis.

In related embodiments there is provided a method for providing anantigen specific immune response to Mycobacterium infection including:

-   -   introducing a strain of Mycobacterium into an antigen-presenting        cell (APC);

wherein said strain includes:

-   -   a recombinant nucleic acid encoding a Mycobacterium antigen for        providing an antigen specific immune response to Mycobacterium        infection;    -   a promoter operably linked to the recombinant nucleic acid for        expression of the recombinant nucleic acid when the strain is        introduced into an APC, said promoter having a sequence of a        Mycobacterium promoter that causes expression of a Mycobacterium        ATP independent chaperone.

In another embodiment there is provided an expression vector including:

-   -   a nucleic acid encoding a Mycobacterium antigen for providing an        antigen specific immune response to Mycobacterium infection;    -   a promoter operably linked to the nucleic acid for expression of        the nucleic acid when the strain is introduced into an APC, said        promoter having a sequence of a Mycobacterium promoter that        causes expression of a Mycobacterium ATP independent chaperone.

In the above described embodiments, the Mycobacterium HspX promoter is apreferred promoter for use as a promoter having the sequence of aMycobacterium promoter that causes expression of a Mycobacterium ATPindependent chaperone.

In another embodiment there is provided a strain of Mycobacteriumincluding the above described expression vector.

In another embodiment there is provided an expression vector including:

-   -   a nucleic acid encoding a protein including a first region        having a sequence encoded by a Mycobacterium CysD gene and a        further region having a sequence encoded by a Mycobacterium        Agb85 gene;    -   a Mycobacterium HspX promoter that causes expression of said        nucleic acid when said vector is introduced into an APC.

In another embodiment there is provided a cell, typically a M.tubercluosis cell or an attenuated Mycobacterium cell, for example anattenuated M. bovis cell such as BCG, or an attenuated M. tuberculosiscell, said cell including the expression vector described above.

In a related embodiment there is provided a method for minimising thelikelihood of development of a M. tubercluosis infection in anindividual including:

-   -   providing an attenuated M. tubercluosis cell in an individual in        which likelihood of development of said infection is to be        minimised;

wherein said cell includes:

an expression vector including:

-   -   a recombinant nucleic acid encoding a protein including a first        region having a sequence encoded by a Mycobacterium Agb85 gene        and a further region having a sequence encoded by a        Mycobacterium CysD gene;    -   a Mycobacterium HspX promoter that causes expression of said        recombinant nucleic acid when said vector is introduced into an        APC;

wherein said cell is provided in said individual in conditions forforming an immune response in the individual to said protein;

thereby minimising the likelihood of development of a Mycobacteriuminfection in the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Up-regulation of the M. tuberculosis sulphate-activation complexwithin host cells and during non-replicating persistence. (A) Geneticorganisation of the cysDNC locus encoding the sulphate activationcomplex. In M. tubercluosis the cysN (GTPase) and cysC (kinase)activities are fused together in one polypeptide and cysDNC constitutean operon. (B) Relative expression level of cysDNC, measured byquantitative real time PCR, of bacilli grown for 48 hours in culture(clear bar) or 48 hours post infection of RAW cells (black bar). Data isthe mean relative expression±S.E.M measured in triplicate and isrepresentative of two independent experiments. (C) Effect of DETA-NO(Det) on growth of cultured M. tuberculosis. 50 μM DETA-NO was added toM. tubercluosis culture (filled squares) every 24 hours for 7 days orleft untreated (filled circles). At days 0, 1, 3 and 7, aliquots weretaken to determine CFU/mL. (D) Relative expression level of cysDNC(black bar) at day 7 post DETA-NO treatment compared to the untreated M.tuberculosis culture (clear bar).

FIG. 2. Host immune recognition of the M. tuberculosissulphate-activation complex. Antigen-specific T cell responses in theMLN of mice were measured 3. (A) and 8 (B) weeks following intranasal M.tubercluosis challenge. IFN-γ secreting cells were enumerated by ELIspotfollowing recall to CysDNC or 85B proteins (10 μg/ml). Data are themeans±S.E.M. for four mice and are representative of duplicateexperiments. The significance of differences between protein-stimulatedand unstimulated cells was determined by ANOVA; *p<0.0001. (C)Antigen-specific T cell responses were measured in the peripheral bloodof M. tuberculosis infected patients (open circles) (n=15) and TST-veindividuals (filled circles) (n=11). T cell proliferation in response toM. tubercluosis CFP, CysDNC and Ag85B proteins at 10 μg/ml was measuredvia the incorporation of ³H-thymidine and a stimulation indexcalculated. Horizontal lines represent the median for each group.Significance of difference's between M. tubercluosis infected patientsand TST-ve individuals was determined by the Mann-Whitney U test;*p<0.001.

FIG. 3. Protection afforded by DNA vaccines -encoding members of thesulphate-activation complex following M. tuberculosis challenge. C57BL/6(n=5) mice were immunised 3 times by i.m injection at 2-week intervalswith 100 μg each of either pCDNA3, DNA-cysD, DNA-cysNC or DNA-cysDcombined with DNA-cysNC. At the time of the first injection of DNAvaccines, mice were immunised once by s.c injection with 5×10⁵ CFU ofBCG. Four weeks following the third immunisation, mice were challengedwith aerosol M. tubercluosis. Four weeks post challenge the bacterialload was determined in the lung (A) and the spleen (B). These data areshown as the mean. CFU (±SEM) per organ and are representative of 1 of 3individual experiments for all groups. The significance of thedifferences between unvaccinated and immunised groups in the lung andspleen were determined by ANOVA; *p<0.001.

FIG. 4. Up-regulation and host immune recognition of downstream enzymesin the M. tuberculosis sulphate-activation pathway. (A) Relativeup-regulation of M. tuberculosis SAP genes within RAW cells compared toin vitro grown bacilli. Total RNA was extracted from in vitro grownbacilli or M. tubercluosis infected RAW cells at 48 hrs post infection 1μg of total RNA was reverse transcribed and subject to real-time PCR toassess expression of M. tubercluosis cysH, sirA, cysK1 and cysE. Data isthe mean relative expression ±S.E.M measured in triplicate and isrepresentative of two independent experiments. (B) Antigen-specific Tcell responses in the MLN of mice were measured 8 weeks following M.tubercluosis challenge. IFN-γ secreting cells were enumerated by ELIspotfollowing recall to CysH, SirA, CysK1 and CysE (10 μg/ml). Data are themeans±S.E.M. for four mice and are representative of duplicateexperiments. The significance of differences between protein-stimulatedand unstimulated cells was determined by ANOVA; *p<0.0001. (C)Recognition of SAP proteins in TB infected individuals. Antigen-specificT cell responses were measured in the peripheral blood of M.tuberculosis infected patients (open circles) (n=15) and TST-veindividuals (filled circles) (n=11). T cell proliferation in response toM. tubercluosis CFP (as per FIG. 2C), CysH, SirA, CysK1 and CysEproteins at 10 μg/ml was measured as described in FIG. 2C. Significanceof differences between M. tubercluosis infected patients and TST-veindividuals was determined by a Mann-Whitney U test; *p<0.001.

FIG. 5. Protective efficacy of DNA vaccines encoding downstream SAPenzymes. C57BL/6 mice (n=5) were immunised 3 times by i.m injection at2-week intervals with 100 μg each of either pCDNA3, DNA-cysD combinedwith DNA-cysNC, or a mix of DNA vaccines expressing cysH, sirA, cysK1 orcysE. At the time of the first injection of DNA vaccines, mice wereimmunised once by s.c injection with 5×10⁵ CFU of BCG. Four weeksfollowing the third immunisation, mice were challenged by the aerosol M.tuberculosis. Four weeks later, the bacterial load was determined in thelung (A) and the spleen (B). These data are shown as the mean CFU (±SEM)per organ and are representative of 1 of 3 individual experiments forall groups. The significance of the differences between unvaccinated andimmunised groups in the lung and spleen (*p<0.0001) and the differencesbetween BCG immunised animals and other immunised groups (+p<0.0001)were determined by ANOVA.

FIG. 6. Protective efficacy of BCG after boosting with CysDN protein.Groups of mice were immunized with 5×105 CFU BCG by s.c injection andafter 24 weeks mice received s.c 100 μg of CysDNC protein in MPL-DDA (3times at 2 week intervals). After 6 weeks mice were challenged withaerosol M. tuberculosis. Control mice were not immunized either noimmunisation or immunisation with BCG and boosted with MPL-DDA adjuvantalone. Four weeks post-challenge the bacterial loads in the lung (A) andspleen (B) were determined. These data are presented as the meanbacterial number ±SEM per organ for 6 to 10 mice per group. Data arerepresentative of 2 independent experiments. The significance ofdifferences between unimmunised mice and other groups (*p<0.0001) and ofthe differences between BCG/MPL-DDA—immunised animals and other groups(**p<0.0001; NS—no significant difference) were determined by ANOVA.

Supplementary FIG. 1. Sulphate assimilation in mycobacteria. Onceimported into the cell, sulphate is activated by ATP Sulfurylase,encoded by cysDNC to produce adenosine-5′-phosphosulfate (APS). Inmycobacteria, APS lies at a metabolic branch point. It could bephosphorylated by APS kianse, encoded by cysC, to producephosphoadenosine-5′-phosphosulfate (PAPS), which functions as theuniversal sulphate donor for the sulfation of biomolecules that are thebuilding block of the mycobacterial cell wall. The sulphate moiety inAPS could also be reduced to sulfite, catalysed by APS reductase andencoded by cysH. Sulfite is further reduced to sulphide by sulfitereductase, (encoded by sirA) and is the form of sulphur that isincorporated into cysteine in the last step of this reductive pathwayinvolving two enzymes, O-Acetylserine sulfhydralse (OASS) and serineacetyltransferase encoded by cysK1 and cysE respectively. Cysteine isthe building block of many important molecules in mycobacteria.

FIG. 7. Rapid induction of the hspX promoter within dendritic cells. Thelevel of GFP expression by BCG Pasteur, aerated BCG:P_(hspX)-GFPcultures (rolling) or BCG:P_(hspX)GFP grown under low oxygen tension for7 days (standing) was measured by flow cytometry (A). BMDC culture fromC57BL/6 mice were infected with BCG:P_(hspX)-GFP at MOI of 1:1 and GFPfluorescence determined at 0, 6 and 24 h post-infection by flowcytometry (B). Data show means±SEM (n=3) and are representative of twoindependent experiments. The significances of differences relative tothe zero timepoint were determined by ANOVA (***p<0.0001). GFPexpression by BCG:P_(hspX)-GFP was visualized by confocal microscopy at0, 6 and 24 h post-BMDC infection (C). Images shown are composites usingthe GFP filter and phase-contrast images to visualize cellularmorphology.

FIG. 8. In vivo induction of the hspX promoter within the host. Micewere vaccinated subcutaneously with 1×10₇ CFU of control BCG orBCG:P_(hspX)-GFP and at 0, 1, 3 and 7 days post-infection the level ofGFP expression in CD45. cells from the infection site was determined(A). Scatter plot of cells from BCG:P_(hspX)-GFP mice (black dots) isoverlaid on the level of GFP expression from mice infected with controlBCG (grey dots). The total number of GFP from the injected is shown in(B). Data shown are the means±SEM and are representative of twoindependent experiments. The significance of differences between day 0and other time-points was determined by ANOVA (**p<0.01; ***p<0.0001).

FIG. 9. Expression of the hspX promoter within dendritic cells recruitedto the site of vaccination. Mice were vaccinated as described in FIG. 2and the number of cells displaying a DC phenotype (CD11c_(hi)CD11b_(hi))was determined by flow cytometry (A). GFP-expressing cells within CD45,population is shown as a scatter plot (B) along with the total number ofGFP CD11b_(hi)CD11c_(hi) cells at the vaccination site over thetimecourse (C). Data shown are means±SEM and are representative of twoindependent experiments. The significance of differences between day 0and other time-points was determined by ANOVA (*p<0.05; **p<0.001;***p<0.0001).

FIG. 10. The hspX promoter can facilitate improved T cell recognition ofa recombinant antigen within APCs. Cultured BMDC were left uninfected(uni) or infected with control BCG, BCG:P_(hspX)-85B or BCG:P_(hspX)-85Bfor 4 h prior to co-incubation with Ag85B-specific T cells purified fromp25 mice. The proliferation (A) and IFN-_ release (B) by p25 T cells atday 3 was determined by [₃H]-thymidine uptake or IFN-_ ELISArespectively. Data show means±SEM (n=3) and are representative of atleast two independent experiments. The significances of differencesbetween uninfected cells and other groups were determined by ANOVA(***p<0.0001; ns, not significant).

FIG. 11. Priming of Ag85B-reactive T cells in the DLNs of micevaccinated with BCG:P_(hspX)-85B. C57BL/6 mice were injectedintravenously with 5×10₅ CFSE-labelled p25 transgenic lymph node cellsand one day later left as uninfected controls (unv) or subcutaneouslyvaccinated with 5×10₅ CFU of BCG, BCG:P_(hsp60)-85B or BCG:P_(hspX)-85B.At 3 or 7 days post-infection, the CFSE and CD62L profile of transferredp25 CD4 T cells in the DLNs was determined (A). The division states arerepresented as CFSE_(hi) (hi), CFSE_(int) (divisions 1-5, int) orCSFE_(lo) (divisions >6, low). The proportion of p25 CD4 T cells indivision states based on CFSE levels was determined (B).

FIG. 12. Increased specific T cell immunity induced by vaccination withBCG expressing Ag85B under the control of the hspX promoter. Mice wereleft unvaccinated (unv) or vaccinated subcutaneoulsy with 5×10₅ CFU ofcontrol BCG, BCG:P_(hsp60)-85B or BCG:P_(hspX)-85B. The number ofIFN-_-secreting splenocytes responding to the p25 peptide from Ag85B wasdetermined at 3 weeks (A) or 12 weeks (B) post-vaccination. Twelve weeksfollowing vaccination, groups of mice were also aerosolized with M.tuberculosis H37Rv and 4 weeks post-challenge the M. tuberculosis loadin the lungs (C) and the spleen (D) were determined. Data shown are themeans±SEM for three mice per group and are representative of twoindependent experiments. The significances of differences betweenunvaccinated mice and BCG-vaccinated groups were determined by ANOVA(*p<0.05; **p<0.001; ***p<0.0001).

FIG. 13. Vaccination with BCG:Ag85BCysD displays improvedimmunogenicity. C57BL/6 mice (n=5) were immunised with 5×10⁵ CFU of BCGor BCG expressing the Ag85BCysD fusion protein. Splenocytesre-stimulated with Ag85BCysD protein demonstrated an increased number ofIFN-g-secreting cells compared with splenocytes from mice immunised withBCG only.

FIG. 14. Protection induced by the Ag85B-CysD fusion protein. C57BL/6mice (n=5) were immunized 3 times by s.c injection with either adjuvant(MPL/DDA), Ag85B-CysD fusion protein (10 mg) or Ag85B (10 mg). At thetime of the first injection of protein vaccines, mice were immunizedonce by s.c injection with 5×10⁵ CFU of BCG. Four weeks following thethird immunization, mice were challenged with aerosol M. tuberculosiswith an infective dose of □100 viable bacilli per lung and bacterialload was determined in the lung (A) and the spleen (B) 4 weeks later.Data are shown as the mean CFU (±SEM) per organ. The significance of thedifferences between groups was evaluated by one-way ANOVA with pair-wisecomparison of multi-grouped data sets achieved using the Bonferroni posthoc test.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

All of the patents and publications referred to herein are incorporatedby reference in their entirety.

For purposes of interpreting this specification, terms used in thesingular will also include the plural and vice versa.

As described herein, the inventors show that sulphate assimilationpathway (SAP) genes and related gene products are highly immunoreactivein the sense that stimulation of lymph node derived lymphocytes from M.tubercluosis-infected mice with SAP proteins provides a strong Th1response lasting for up to 8 weeks post infection. Further, in humansinfected with M. tuberculosis, peripheral blood lymphocytes wereobserved to proliferate in response to SAP proteins to a greater extentthan cells from M. tubercluosis negative individuals.

Further to the above, the inventors show that SAP genes and proteins canbe utilised to induce an antigen specific protective immune response.More specifically, the inventors show that immunisation with SAP genesand proteins provides protection to lungs and spleen in micesubsequently infected with M. tubercluosis and the protective efficacyapproached that observed with BCG immunisation.

Still further, the inventors show that SAP genes and gene products canbe used to improve the protective immunity provided by BCG vaccinationwhereby, as shown in the specification, the SAP components improved theprotective effect of BCG against M. tubercluosis infection which wasmost apparent in lung tissue.

As discussed herein, these findings are unanticipated as SAP proteinsare proposed to be intracellular or membrane associated proteins which,given location, should be less likely than secreted proteins to besubject to immune surveillance. In this regard it is surprising that theamount of immune activation observed in these studies is equivalent tothat observed for the cell surface antigen or secreted antigen, Ag85B.

A. Definitions

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

Sulphate assimilation pathway or SAP generally refers to the pathway bywhich Mycobacteria reduce sulphur, thereby obtaining substrate for thebiosynthesis of cysteine and downstream products including mycothiol. Inmore detail, the pathway involves the formation of adenosine5′-phosphosulfate (APS) from sulphate, from which APS reductase (encodedby CysH) may produce sulphite, and from which sulfite reductase (encodedby SirA) may produce sulphide, and from which, and withO-Acetyl-L-serine, O-Acetyl-L-Serine Sulfhydalase (encoded by CysK1) mayproduce cysteine. Key enzymes of SAP include ATP sulfurylase(adenylyl-transferase) (encoded by CysD), GTPase (encoded by CysN), andAPS kinase. These enzymes enable the formation of PAS from sulphate, andthe formation of 3′ phosphoadenosine 5′-phosphosulfate (PAPS) from APS.

A SAP component generally refers to a protein or enzyme involved in thereduction of sulphur in Mycobacterium according to the SAP, examples ofwhich include those encoded by CysD, CysNC, CysH, SirA, CysE, CysK1genes.

Sulphate activating complex or SAC generally refers to a heterodimericcomplex formed from the association of the CysD gene product with theCysNC gene product. The CysD and CysNC genes exist together inMycobacteria as an operon.

CysD gene generally refers to a nucleic acid encoding a ATP sulfurylase.The nucleic acid may have a nucleotide sequence substantially as shownin SEQ ID No: 1 herein or otherwise having defined homology and/oridentity as defined herein.

CysD protein generally refers to a ATP sulfurylase. The protein may havean amino acid sequence substantially as shown in SEQ ID No: 2 herein orotherwise having defined homology and/or identity as defined herein.

CysNC gene generally refers to a nucleic acid encoding a GTPase and APSkinase. The nucleic acid may have a nucleotide sequence substantially asshown in SEQ ID No: 3 herein or otherwise having defined homology and/oridentity as defined herein.

CysNC protein generally refers to a GTPase and APS kinase. The proteinmay have an amino acid sequence substantially as shown in SEQ ID No: 4herein or otherwise having defined homology and/or identity as definedherein.

Ag85B gene generally refers to a nucleic acid having a nucleotidesequence substantially as shown in SEQ ID No: 5 herein or otherwisehaving defined homology and/or identity as defined herein.

Ag85B protein generally refers to a protein having an amino acidsequence substantially as shown in SEQ ID No: 6 herein or otherwisehaving defined homology and/or identity as defined herein.

HspX promoter generally refers to a nucleic acid having a nucleotidesequence substantially as shown in SEQ ID No: 7 herein or otherwisehaving defined homology and/or identity as defined herein.

85BCysD generally refers to a protein having an amino acid sequencesubstantially as shown in SEQ ID No: 8 herein.

pHspX85BCysD generally refers to a nucleic acid having a nucleotidesequence substantially as shown in SEQ ID No: 9 herein.

B. Induction of Antigen Specific Immunity

As discussed herein, and exemplified in the examples, the inventors haveshown that SAP components elicit an antigen specific protective immuneresponse. Specifically, immunisation with SAP components, and inparticular, SAC components prevents the progression of Mycobacteriuminfection in mice later infected with M. tuberculosis. Further, theinventors show that the SAP components can be used to boost immunityarising from BCG immunisation, and that the SAP components are highlyimmunoreactive in M. tubercluosis infected individuals, suggesting thatthese components are useful for minimising development of a disease orcondition caused by infection. Therefore, the invention provides methodsfor: (i) prophylaxis; (ii) treatment; and (iii) boosting immunity toMycobacterium infection. It is in these contexts that the methods of theinvention minimise the likelihood of development of an infection, eitherby preventing the infection from developing to a relevant disease orpathology, or by preventing further development of a disease orpathology once an infection has been established.

Accordingly in one embodiment there is provided a method for minimisingthe likelihood of development of a Mycobacterium infection in anindividual including:

-   -   forming an immune response to a component of a Mycobacterium        sulphate assimilation pathway (SAP) in an individual;

thereby minimising the likelihood of a Mycobacterium infection fromdeveloping in the individual.

In one embodiment, the individual may not have a detectableMycobacterium infection and/or may not have been previously immunisedagainst Mycobacterium. Such an individual can generally be identified bythe Mantoux test which is widely used in the art.

In another embodiment, the individual may be asymptomatic or havesub-clinical symptoms of infection. An asymptomatic subject moretypically, has one or more symptoms (e.g., fever, cough, weight loss).Bacilli may be present and culturable, i.e., can be grown in culturefrom the above body fluids and individuals may have radiographicallyevident pulmonary lesions which may include infiltration but withoutcavitation.

In another embodiment the individual may have obvious symptoms ofinfection such as cavitary lesions in the lungs. Bacilli may beculturable from smears of sputum and/or the other body fluids notedabove, but also present in sufficient numbers to be detectable asacid-fast bacilli in smears of these fluids.

Typically the immune response is predominantly a Th1 response. Thisresponse is determined by detecting cellular proliferation afteradministration of the vaccine as measured by ³H thymidine incorporation,or using cellular assays in which IFN-γ production is assessed, such asflow cytometry and/or ELISA. The immune response can also be measured bydetecting specific antibodies (at a titer in the range of 1 to 1×10⁶,preferably 1×10³, more preferable in the range of about 1×10³ to about1×10⁶, and most preferably greater than 1×10⁶).

An in vitro cellular response is determined by release of a relevantcytokine such as IFN-gamma, from lymphocytes withdrawn from an animal orhuman currently or previously infected with virulent mycobacteria, or bydetection of proliferation of these T cells. The induction is performedby addition of the polypeptide or the immunogenic portion to asuspension comprising from 1×10⁵ cells to 3×10⁵ cells per well. Thecells are isolated from either blood, the spleen, the liver or the lungand the addition of the polypeptide or the immunogenic portion of thepolypeptide result in a concentration of not more than 20 ug per mlsuspension and the stimulation is performed from two to five days. Formonitoring cell proliferation the cells are pulsed with radioactivelabelled thymidine and after 16-22 hours of incubation the proliferationis detected by liquid scintillation counting. A positive response is aresponse more than background plus two standard deviations. The releaseof IFN-gamma can be determined by the ELISA method, which is well knownto a person skilled in the art. A positive response is a response morethan background plus two standard deviations. Other cytokines thanIFN-gamma could be relevant when monitoring an immunological response tothe polypeptide, such as IL-12, TNF-.alpha., IL-4, IL-5, IL-10, IL-6,TGF-.beta. Another and more sensitive method for determining thepresence of a cytokine (e.g. IFN-gamma) is the ELISPOT method where thecells isolated from either the blood, the spleen, the liver or the lungare diluted to a concentration of preferable of 1 to 4×10⁶ cells/ml andincubated for 18-22 hrs in the presence of the polypeptide or theimmunogenic portion of the polypeptide resulting in a concentration ofnot more than 20 ug per ml. The cell suspensions are hereafter dilutedto 1 to 2×10⁶/ml and transferred to Maxisorp plates coated withanti-IFN-gamma and incubated for preferably 4 to 16 hours. The IFN-gammaproducing cells are determined by the use of labelled secondaryanti-IFN-antibody and a relevant substrate giving rise to spots, whichcan be enumerated using a dissection microscope. It is also apossibility to determine the presence of mRNA coding for the relevantcytokine by the use of the PCR technique. Usually one or more cytokineswill be measured utilizing for example the PCR, ELISPOT or ELISA. Itwill be appreciated by a person skilled in the art that a significantincrease or decrease in the amount of any of these cytokines induced bya specific polypeptide can be used in evaluation of the immunologicalactivity of the polypeptide.

The immune response may be formed by providing a component of aMycobacterium SAP in an individual in conditions for enabling formationof an immune response to said component in said individual. Thesecomponents may be provided in various forms as discussed under therelevant subheadings below.

Recombinant BCG and other live mycobacterium can be deliveredsubcutaneously or by inhalation. The dosage regimen may also bedetermined by the skilled person using his expertise (e.g. singleadministration, repeated administration (twice or more at regular orirregular intervals), etc. This will typically also depend on thedisease to be treated and the individual receiving the treatment (inbladder cancer in humans, for instance, a low-dose BCG regimen has beendescribed as 75 mg, while a standard dose is 150 mg). However, doses ofBCG as low as 1 mg have been documented to effectively support an immuneresponse for a long period of time (5 years). In tuberculosis, anexample of a typical dose is much lower: 0.075 mg, corresponding to0.3-1.2 million living mycobacteria). Roughly speaking, a typical dosemay fall between 0.01 μg/kg body weight and 10 mg/kg body weight. Intreatment of tuberculosis, one treatment typically protects for a numberof years. However, it is also envisaged that repeat doses are given (asis e.g. typically the case in treatment of bladder cancer).

Where the vaccine or immune stimulating composition is peptide based,the manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These includeoral, nasal or mucosal application in either a solid form containing theactive ingredients (such as a pill, suppository or capsule) or in aphysiologically acceptable dispersion, such as a spray, powder orliquid, or parenterally, by injection, for example, subcutaneously,intradermally or intramuscularly or transdermally applied. The dosage ofthe vaccine will depend on the route of administration and will varyaccording to the age of the person to be vaccinated and, to a lesserdegree, the size of the person to be vaccinated. Currently, mostvaccines are administered intramuscularly by needle injection and thisis likely to continue as the standard route. However, vaccineformulations which induce mucosal immunity have been developed,typically by oral or nasal delivery. One of the most widely studieddelivery systems for induction of mucosal immunity contains choleratoxin (CT) or its B subunit. This protein enhances mucosal immuneresponses and induces IgA production when administered in vaccineformulations. An advantage is the ease of delivery of oral or nasalvaccines. Modified toxins from other microbial species, which havereduced toxicity but retained immunostimulatory capacity, such asmodified heat-labile toxin from Gram-negative bacteria or staphylococcalenterotoxins may also be used to generate a similar effect. Thesemolecules are particularly suited to mucosal administration.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and advantageously contain 10-95% of activeingredient, preferably 25-70%.

Where the vaccine or immune stimulating composition is a viral vector, acarrier can be any substance that does not itself induce the productionof antibodies harmful to the patient receiving the composition, andwhich can be administered without undue toxicity. Suitable carriers canbe large, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Suchcarriers are well known to those of ordinary skill in the art.Pharmaceutically acceptable carriers can include liquids such as water,saline, glycerol and ethanol. Auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, can also bepresent in such vehicles. Stabilizing agents such as trehalose orsubstances that allow water-soluble sugar glass formation at ambienttemperatures may also be present. The latter includes the use of mixedsoluble glass stabilisation technology in microsphere format suspendedin perfluorocarbon liquids. Liposomes are also suitable carriers. Athorough discussion of pharmaceutical carriers is available in Gennaro(2000) Remington: The Science and Practice of Pharmacy. 20th ed., ISBN:0683306472. The pharmaceutical composition is preferably sterile. It ispreferably pyrogen-free. It is preferably buffered e.g. at between pH 6and pH 8, generally around pH 7. Preferably, the composition issubstantially isotonic with humans. The compositions of the inventionmay be administered via a variety of different routes. Certain routesmay be favoured for certain compositions, as resulting in the generationof a more effective response, or as being less likely to induce sideeffects, or as being easier for administration. For example, thecompositions utilised in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal or transcutaneous applications,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,intravaginal or rectal means. The compositions may be prepared forintranasal administration, as nasal spray, nasal drops, gel or powder,as injectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. Direct delivery of the compositions willgenerally be accomplished by injection, subcutaneously,intraperitoneally, intravenously or intramuscularly, intranasally, ordelivered to the interstitial space of a tissue. Dosage treatment may bea single dose schedule or a multiple dose schedule.

C. Determining Immunity to Mycobacterium Infection

In another embodiment there is provided a method for determining whetheran individual is immune to a Mycobacterium including:

-   -   providing a component of a Mycobacterium SAP in an individual in        conditions for enabling formation of an immune response to said        component in said individual;    -   determining whether the individual develops a protective immune        response to said component;

wherein development of a protective immune response determines that theindividual is immune to a Mycobacterium;

thereby determining whether the individual is immune to a Mycobacterium.

The method is particularly useful to confirm the result of Mantouxtesting, or other conventional Mycobacterium testing, or otherwise forfurther delineating a Mantoux test result, for example to identify keyspecificities in a particular immune response, or nature of the relevantimmunogens on which the response is based. The various assays mentionedin the previous sub-heading for detecting formation of an immuneresponse can be implemented here.

D. Vaccines and Immune Stimulating Compositions

The invention provides vaccines and immune stimulating compositions. Incertain embodiments the vaccines and immune stimulating compositions areuseful for providing a protective immune response to Mycobacteriuminfection, especially M. tubercluosis infection.

Generally, four different forms of vaccine or composition are describedas follows:

(i) those that are acellular and that contain as an active ingredientfor immune stimulation, a recombinant or synthetic SAP component.

(ii) those that contain as an active ingredient for immune stimulation,a cellular extract that may be enriched for a SAP component.

(iii) those that contain as an active ingredient for immune stimulation,a cell that expresses a recombinant SAP component.

(iv) those that contain a nucleic acid encoding a SAP component that onadministration to an individual is expressed to form the activeingredient for immune stimulation.

(v) those that contain a viral vector encoding a SAP component thatforms an active ingredient for immunisation.

A preferred immunogen in each of these forms is the CysD gene or geneproduct, or fragments of homologs thereof.

These forms are described in more detail below.

D.1 Acellular Compositions Containing a Recombinant or Synthetic SAPComponent.

In one embodiment, the immune response is formed by providing acomponent of the SAP in the form of an acellular composition includingisolated or recombinant SAP protein in the individual.

Generally these compositions include two key components, the first beingthe immunogen in the form of the recombinant or synthetic SAP componentand the second being an adjuvant for potentiating an immune response tothe immunogen.

Turning to the immunogen, this may include any one or more of the SAPcomponents as described herein. Preferably the immunogen includes CysD,or an immunogenic or antigenic fragment or homolog thereof. Fragmentsand homologs are described in more detail below.

It will be understood that the immunogen may further include otherrecombinant or synthetic Mycobacterium antigens or immunogens. These maybe provided in a form whereby they are fused to CysD by a covalent bond,where they are non covalently associated with CysD, or where they arenot bound to CysD at all. Particular examples of these antigens andimmunogens include Ag85B. Others are discussed in WO2009/070700.

In one embodiment, a given SAP component (such as CysD, CysNC, CysH,SirA, CysE, CysK1 proteins and their encoding genes) may have aconserved function in terms of activity in the sulphate assimilationpathway and yet have a diverged sequence. These proteins or nucleicacids are referred to as homologs.

In certain embodiments a given SAP component is one having at least 75%,preferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 98% or 99% identity to a given SAPcomponent. For example a CysD immunogen may be one having at least 75%,preferably 80%, more preferably 85%, more preferably 90%, morepreferably 95%, more preferably 98% or 99% identity to a CysD proteinshown in SEQ ID No: 2. The nucleic acid sequence encoding the CysDimmunogen may be one having at least 75%, preferably 80%, morepreferably 85%, more preferably 90%, more preferably 95%, morepreferably 98% or 99% identity to a CysD gene shown in SEQ ID No: 1.

Percent sequence identity is determined by conventional methods, bymeans of computer programs known in the art such as GAP provided in theGCG program package (Program Manual for the Wisconsin Package, Version8, August 1994, Genetics Computer Group, 575 Science Drive, Madison,Wis., USA 53711) as disclosed in Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48, 443-453, which is herebyincorporated by reference in its entirety. GAP is used with thefollowing settings for polypeptide sequence comparison: GAP creationpenalty of 3.0 and GAP extension penalty of 0.1. Sequence identity ofpolynucleotide molecules is determined by similar methods using GAP withthe following settings for DNA sequence comparison: GAP creation penaltyof 5.0 and GAP extension penalty of 0.3.

In another embodiment a given SAP component is provided in the form of afragment that is capable of forming a protective immune response. Thesefragments are generally of sufficient length and conformation enablingpresentation by the APC on Class I or II. They may be of a lengthranging generally from about 8 to 15, and about 8 to 11 for Class Ipresentation and 11 to 15 for Class II presentation.

In order to identify relevant T-cell epitopes which are recognizedduring an immune response, a common method is to use overlappingpeptides for the detection of MHC class II epitopes, preferablysynthetic, having a length of e.g. 20 amino acid residues derived fromthe polypeptide. These peptides can be tested in biological assays, suchas the IFN-gamma assay as described herein and those that give apositive response will be classed as immunogenic T cell epitopes. MHCclass I epitopes can be identified by predicting those peptides thatwill bind to Class I (Stryhn, A., et al 1996 Eur. J. Immunol.26:1911-1918) and hereafter produce these peptides synthetically andtest them in relevant biological assays e.g. the IFN-gamma assay asdescribed herein.

The peptides preferably having a length of e.g. 8 to 11 amino acidresidues derived from the polypeptide. B-cell epitopes can be determinedby analyzing the B cell recognition to overlapping peptides covering thepolypeptide of interest as e.g. described in Harboe, M., et al 1998Infect. Immun. 66:2; 717-723

Any of CysD, CysNC, CysH, SirA, CysE, CysK1 or related nucleic acidsequences can be made by solid phase synthesis or recombinant DNAtechnology.

Turning to the adjuvant, there are many examples of adjuvants known inthe art. See also Allison (1998, Dev. Biol. Stand., 92:3-11;incorporated herein by reference), Unkeless et al. (1998, Annu. Rev.Immunol., 6:251-281), and Phillips et al. (1992, Vaccine, 10:151-158).Exemplary adjuvants that can be utilized in accordance with theinvention include, but are not limited to, cytokines, aluminium salts(e.g., aluminium hydroxide, aluminium phosphate, etc.; Baylor et al.,Vaccine, 20:S18, 2002), gel-type adjuvants (e.g., calcium phosphate,etc.); microbial adjuvants (e.g., immunomodulatory DNA sequences thatinclude CpG motifs; endotoxins such as monophosphoryl lipid A (Ribi etal., 1986, Immunology and Immunopharmacology of bacterial endotoxins,Plenum Publ. Corp., NY, p407, 1986); exotoxins such as cholera toxin, E.coli heat labile toxin, and pertussis toxin; muramyl dipeptide, etc.);oil-emulsion and emulsifier-based adjuvants (e.g., Freund's Adjuvant,MF59 [Novartis], SAF, etc.); particulate adjuvants (e.g., liposomes,biodegradable microspheres, etc.); synthetic adjuvants (e.g., nonionicblock copolymers, muramyl peptide analogues, polyphosphazene, syntheticpolynucleotides, etc.); and/or combinations thereof. Other exemplaryadjuvants include some polymers (e.g., polyphosphazenes; described inU.S. Pat. No. 5,500,161), Q57, saponins (e.g., QS21, Ghochikyan et al.,Vaccine, 24:2275, 2006), squalene, tetrachlorodecaoxide, CPG 7909(Cooper et al., Vaccine, 22:3136, 2004),poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., Vaccine,16:92, 1998), interferon-γ (Cao et al., Vaccine, 10:238, 1992), blockcopolymer P1205 (CRL1005; Katz et al., Vaccine, 18:2177, 2000),interleukin-2 (IL-2; Mbwuike et al., Vaccine, 8:347, 1990), polymethylmethacrylate (PMMA; Kreuter et al., J. Pharm. ScL, 70:367, 1981),dimethyloctadecylammonium bromide (DDA), IC31® (Vann Dissel, Vaccine,29:2100, 2011), etc.

These compositions may also include diluents, excipients and carriersenabling administration of the composition, as known in the art.

D.2 Cell Extracts

In another embodiment, the immune response is formed by providing acomponent of the SAP in the form of a cell extract including an isolatedor recombinant SAP protein in the individual.

Cell extracts may be obtained by known techniques, including for examplesonification of a Mycobacterium strain, pelleting by centrifugation andretrieving lysate for immunisation. The lysate could be further enrichedfor SAP components by for example immunoaffinity chromatography. This ismost useful where the strain is not recombinant for a SAP component,i.e. where the strain is not otherwise an over-expressos of, or enrichedfor, a SAP component. Where the strain is a recombinant strain havinghigh levels of expression of a SAP component, a chromatographicenrichment step may not be necessary.

The cell extracts may be particularly useful as an in vitro reagent fortesting efficacy of an immunisation protocol.

D.3 Recombinant or Transformed Cells

In one embodiment of the invention, the immune response is formed byproviding a component of the SAP in the form of a cell including anisolated or recombinant SAP protein in the individual.

It is particularly preferred that the cell is a Mycobacterium, and inparticular a M. tubercluosis strain, especially an attenuated M.tuberculosis strain capable of forming a live attenuated vaccine. A‘live attenuated vaccine’ as used herein is a vaccine containing live orviable micro-organisms with reduced virulence (attenuated); as opposedto an inactivated vaccine.

Other types of mycobacterium include members of the Mycobacteriumtuberculosis complex (Taxon identifier 77643 in the UniProt or NCBItaxonomy database), which includes the species M. tuberculosis (themajor cause of human tuberculosis), M. bovis, M. bovis BCG (the strainmost often used for vaccination purposes), M. africanum, M. microti, M.canetti, and M. pinnipedii.

Preferably the cell is a BCG strain. Those of skill in the art willrecognize that several suitable BCG strains exist which are suitable foruse in the practice of the invention, including but not limited to thosedesignated ATCC® Number:

27289 Mycobacterium bovis BCG, Chicago 1 [B; BCGT; tice]

27291 M. bovis

35731 M. bovis TMC 1002 [BCG Birkhaug]

35732 M. bovis TMC 1009 [BCG Swedish]

35735 M. bovis TMC 1012 [BCG Montreal; CIP 105920]

35736 M. bovis TMC 1013 [BCG Brazilian]

35737 M. bovis TMC 1019 [BCG Japanese]

35738 M. bovis TMC 1020 [BCG Mexican]

35739 M. bovis TMC 1021 [BCG Australian]

35741 M. bovis [BCG Glaxo]

35742 M. bovis TMC 1025 [BCG Prague]

35744 TMC 1029 [BCG Phipps]

35745 M. bovis TMC 1030 [BCG Connaught]

35746 M. bovis TMC 1101 [BCG Montreal, SM-R]

35747 M. bovis TMC 1103 [BCG Montreal, INH-R; CIP 105919]

35748 M. bovis TMC 1108 [BCG Pasteur SM-R]

27290 M. bovis [BCG Copenhagen H]

19274 M. bovis deposited as zrculosis subspecies bovis 50 [BCG]

19015 Mycobacterium sp. d<i as M. bovis Karlson and Lessel BCG

35733 M. bovis TMC 1010 [BCG Danish, SSI 1331] and

35734 M. bovis TMC 1011 [BCG Pasteur], etc.

It will be understood that the. recombinant mycobacteria of theinvention need not be confined to strains of BCG. Those of skill in theart will recognize that other Mycobacterium strains may also beemployed, examples of which include but are not limited to: M.tuberculosis CDC1551 strain (See, e.g. Griffith et at., Am. J. Respir.Crit. Care Med. August;152(2):808; 1995), M. tuberculosis Beijing strain(van Soolingen et al., J CHn Microbiol. December:33(12):3234-8,1995)H37Rv strain (ATCC#:25618), M. tuberculosis pantothenate auxotrophstrain (Sambandamurthy et al., supra; M. tuberculosis rpo V mutantstrain (Collins et al., Proc Natl Acad Sci USA. 92(17):8036; 1995), M.tuberculosis leucine auxotroph strain (Hondalus et al., Infect. Immun.68(5):2888; 2000), etc., or other attenuated and/or recombinant strainsderived from M. tuberculosis. Other candidate bacteria include othermembers of the M. tuberculosis complex, other mycobacteria (e.g. M.africanum or M. avium complex bacteria), or other mycobacterial species.

In this form of the invention, SAP component may be over-expressed, i.e.the protein may be expressed at a level that exceeds that of a suitablecontrol organism, such as the same mycobacterium that has not beengenetically engineered to over-express the SAP component. Those of skillin the art are well acquainted with comparative measurements of proteinactivity, and with the use of suitable standards and controls for suchmeasurements.

The over-expression of the SAP component may be carried out by anysuitable method known in the art. Generally, the method will involvelinking nucleic acid sequences encoding the SAP component to aparticular promoter or other regulatory element that is activated whenthe strain is introduced into a cell especially an APC. Those of skillin the art will recognize that many such expression control sequencesare known and would be suitable for use in the invention. For example,if constitutive expression of SAP component is desired, expressioncontrol sequences (e.g. promoters and associated sequences) includingbut not limited to: mycobacterium optimal promoter (mop, George et al.,1995); blaF promoter (Timm et al., 1994); hsp60, ace or msp12 promoters;etc., with or without an optimized ribosomal binding site.

Alternatively, over-expression of SAP may not be constitutive but mayinstead be inducible, in response to an environmental cue. For example,expression of the protein may be driven by a promoter that is induced ina particular location or in response to an environmental stimulus,examples of which include but are not limited to: macrophage induciblepromoter (which drives expression of genes that are specificallyupregulated within the macrophage phagosome, see Schannapinger et al.JEM 2003); acetamidase promoter (Mahenthiralingam et al., J. Gen.Microbiol. 1993), and tetracycline-inducible (Blokpoel et al., Nucl.Acids Res. 33(2):e22, 2005), etc.

In addition, promoters from other species maybe utilized, examples ofwhich include but are not limited to: various viral promoters, wherebyafter “gene therapy-like” strategies (e.g. co-inoculation ofmycobacteria, and an engineered virus) the Mtb antigens are expressed inselected tissues infected by the co-administered virus; etc.

As a further alternative, native or naturally occurring SAP promotersmay be altered by mutation to cause over-expression of SAP component.

Those of skill in the art will recognize that several avenues areavailable to introduce nucleic acid sequences encoding the SAPcomponent, in operable linkage with one or more expression controlsequences, into a mycobacterial host where over-expression will occur.For example, the sequences may be included in a vector that issubsequently introduced into the mycobacterium. Many vectors suitablefor containing and expressing genes are known, and include but are notlimited to various extra-chromosomal elements such as plasmids, e.g.those comprising the pAL500 origin of replication, modified to augmenttheir copy number; or other plasmids with origins of replication thatare or will be developed; or extrachromosomal elements that do notreplicate or integrate into mycobacterial genome but provide a suicidalsource for homologous recombination to occur; etc. Introduction of sucha vector into a mycobacterium may be carried out by any of several knownmethods suitable for that particular vector, including but not limitedto electroporation and mycobacteriophage-mediated transduction forhomologous recombination. In a preferred embodiment, the vector is aplasmid and the method that is used is electroporation.

In other embodiments of the invention, the SAP component isover-expressed from the M. tuberculosis chromosome. Those of skill inthe art will recognize that various molecular biology strategies existfor generating a mycobacterium with this property. For example, variousmutations may be introduced into the chromosome (randomly or in adirected fashion) that result in over-production of the SAP component bythe bacterium. Alternatively, nucleic acid sequences that include one ormore expression control sequences operably linked to nucleic acidsequences encoding the SAP component may be introduced into thebacterial chromosome, e.g. by transduction with a suicide plasmid withor without a means for counter-selection, to provide sequences forhomologous recombination.

In a particularly preferred embodiment of the invention, there isprovided an expression vector including:

-   -   a nucleic acid encoding a Mycobacterium antigen for providing an        antigen specific immune response to Mycobacterium infection;    -   a promoter operably linked to the nucleic acid for expression of        the nucleic acid when the strain is introduced into an APC, said        promoter having a sequence of a Mycobacterium promoter that        causes immediate and sustained expression of a Mycobacterium        antigen when said promoter is introduced into an APC.

In the above described embodiments, the Mycobacterium HspX promoter is apreferred promoter for use as a promoter having the sequence of aMycobacterium promoter that causes expression of a Mycobacterium ATPindependent chaperone. Specifically, as exemplified in Example 2 here,this promoter is capable of inducing high and sustained expression of aSAP component in a BCG strain. Other examples of promoters useful in theinvention include Rv0962c. Rv0971c. Rv0983. Rv0986.Rv2428.Rv1130.Rv2626c as described in Fontan et al. 2008 Infect. &Immun. 76:pp 717.

Vaccine formulation under this subheading involves studies to determinemaximum bacterial viability and stability throughout the manufacturingprocess. This includes determination of maximum organism viability (liveto dead) during culture utilizing a variety of commonly used medium forthe culture of Mycobacteria to include the addition of glycerol, sugars,amino acids, and detergents or salts. After culture cells are harvestedby centrifugation or tangential flow filtration and resuspended in astabilizing medium that allows for protection of cells during freezingor freeze-drying process. Commonly used stabilizing agents includesodium glutamate, amino acids or amino acid derivatives, glycerol,sugars or commonly used salts. The final formulation will providesufficient viable organisms to be delivered by intradermal, percutaneousinjection, perfusion or oral delivery with sufficient stability tomaintain and adequate shelf life for distribution and use.

Prior to administration to humans as a vaccine, the BCG strainsdescribed under this subheading are tested according to methods that arewell-known to those of skill in the art. For example, tests fortoxicity, virulence, safety, etc. are carried out in suitable animalmodels, e.g. in mice, guinea pigs, etc., some of which areimmunocompromised. The ability of the vaccine preparations to elicit animmune response is likewise typically tested in suitable animal models,e.g. mice, guinea pigs, etc. In addition, protection studies involvingvaccination, boosting, and subsequent challenge with live Mtb may becarried out using suitable animal models such as mice, guinea pigs, andnon-human primates. Finally, those of skill in the art are familiar withthe arrangements for carrying out clinical trials in consenting humans,in order to test the efficacy of the vaccine preparations. For details,see, for example, United States patent application 20060121054 (Sun etal.) published Jun. 8, 2006, and references cited therein.

D.4 Nucleic Acid Vaccines

In another embodiment, the immune response is formed by providing acomponent of the SAP in the form of or a nucleic acid encoding a SAPprotein in the individual.

In particular, as exemplified Example 1, the inventors have shown thatprotective immunity can be provided by administering a DNA vaccinecontaining a gene encoding CysD in mice challenged with M. tubercluosis.

The nucleic acid may be provided in linearised or circular form forinjection. Generally the nucleic acid will have a promoter enablingexpression of the SAP component in the relevant cell. For example, wherethe administration is to muscle tissue, the vector will contain apromoter capable of activation by muscle transcription factors andenhancers. In these embodiments, it is generally understood that themuscle cell will produce the relevant SAP component which will then bephagocytosed by an APC such as a dendritic cell for presentation to a Tcell, upon which immunity is established.

D.5 Viral Vectors

In another embodiment, the immune response is formed by providing acomponent of the SAP in the form of a viral vector that contains anucleic acid that encodes the component, or that expresses a componentof the SAP.

Examples of suitable vectors include those based on a vaccinia genomeand those based on an adenovirus genome.

Some of the compositions described under the above subheadings may beformulated as liquid solutions or suspensions, however solid forms suchas tablets, pills, powders and the like are also contemplated. Solidforms suitable for solution in, or suspension in, liquids prior toadministration may also be prepared. The preparation may also beemulsified. The active ingredients may be mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredients. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol and the like, or combinations thereof. Inaddition, the composition may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and the like. If it is desired to administer an oral form of thecomposition, various thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders and the like may be added. The composition ofthe present invention may contain any such additional ingredients so asto provide the composition in a form suitable for administration.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

EXAMPLES Example 1

M. tuberculosis can survive in a broad spectrum of environments,including high levels of oxidative stress, low pH and nutrientdeprivation ((Nathan, 2000 #29). Exposure and adaption of M.tuberculosis to these conditions during infection requires thecoordinated regulation of gene expression ((Timm, 2003 #93). Genesinvolved in the metabolism of sulphur have consistently been identifiedas up-regulated in conditions that mimic the macrophage environment((Pinto, 2004 #14;, #35;Muttucumaru, 2004 #34;Manganelli, 2002#75;Hampshire, 2004 #33;Betts, 2002 #49) and during macrophage infection((Schnappinger, 2003 #31). These genes encode enzymes of the sulphateassimilation pathway (SAP) of M. tubercluosis, required for thereduction of sulphur. Indeed, sulphur-containing compounds arefundamental in a wide range of biological activities. In its reducedform sulphur is used in the biosynthesis of the amino acid cysteine, oneof the prime targets for reactive nitrogen intermediates encountered byM. tubercluosis in the intracellular environment ((Rhee, 2005 #92).Cysteine can be subsequently incorporated into mycothiol which functionsanalogously to glutathione ((Fan, 2009 #197) and is crucial to M.tubercluosis within the granuloma in regulating the redox balance uponencountering free radicals released by host cells. Mutants ofMycobacterium smegmatis in which mycothiol biosynthesis has beenabrogated exhibit high-level resistance to isoniazid and are moresusceptible than wild-type strains to oxidative stress and antibiotics((Rawat, 2002 #82;Buchmeier, 2003 #85). This first line of defence by M.tuberculosis therefore is linked to the availability of cysteine and assuch has been shown to be required for the organism's surivial ((Sareen,2003 #84;Buchmeier, 2006 #83;Newton, 2002 #81). Reinforcing thisincreased need for cysteine in the macrophage environment, is theup-regulation of ATP sulfurylase, the first enzyme in the SAP, uponexposure to oxidative stress ((Pinto, 2004 #14;, #35;Schnappinger, 2003#31). Disabling the biosynthesis of cysteine attenuates bacterialvirulence and persistence during the chronic phase of infection in mice((Senaratne, 2006 #3).

Although members of the SAP appear to be highly expressed underconditions presumably encountered within the host, it is not known ifthese proteins constitute immunogenic components of M. tuberculosis.Most focus has been on secreted proteins of mycobacteria, as these arepredicted to be recognized by early host immune responses ((Andersen,1992 #202;Roberts, 1995 #203). Sulphate reduction takes place within thecell and for this reason SAP enzymes are either intracellular ormembrane bound components ((de Souza, 2011 #206;Bhave, 2007#205;Schelle, 2006 #204) and would not be detected in screens forimmunogenic secreted antigens of M. tubercluosis ((Andersen, 1992 #202).In this report we demonstrate that members of the SAP are highlyimmunogenic components of M. tubercluosis, being recognised by M.tubercluosis infected individuals and conferring protective immunity ina murine model of TB. Our results suggest that SAP members are potentialcandidates for inclusion into new TB vaccines.

Materials and Methods

Bacterial Strains and Growth Conditions.

Escherichia coli K-12 and BL21 (DE3) were grown in Luria-Bertani (LB)broth or agar (Sigma-Aldrich). M. tubercluosis H37Rv or strain MT103((Jackson, 1999 #208) were grown in Middlebrook 7H9 medium (DifcoLaboratories) supplemented with 0.5% glycerol, 0.05% Tween 80, and 10%albumin-dextrose-catalase (ADC) or on solid Middlebrook 7H11 medium(Difco Laboratories) supplemented with oleic acid—ADC. All cultures weregrown at 37° C. with or without shaking. Antibiotics were added to mediawhen required at 25 μg/mL for kanamycin and 100 μg/mL for ampicillin.Non-replicating persistence of in vitro grown M. tubercluosis wasachieved by adding daily 50 μM of the nitric oxide-donating compound2,2′-(Hydroxynitrosohydrazono)bis-ethanimine (DETA-NO) (Sigma) using themethod of ((Bryk, 2008 #36). At day 0, 1, 3 and 7 bacterial counts weredetermined for these cultures and at day 7, RNA was extracted from bothcultures for real-time (RT) PCR analysis.

Protein Antigens and DNA Vaccines

Culture Filtrate protein (CFP) was obtained from the NIH Biodefense andEmerging Infections Research Resources Repository (NR-14825). ConcavalinA (ConA) was purchased from Sigma-Aldrich. Purification of SAP proteinantigens and construction of DNA vectors encoding SAP genes aredescribed in supplementary table 1.

Macrophage Infection and Real-Time PCR

The RAW264.7 mouse macrophage cell line was grown in RPMI (Gibco-BRL)supplemented with 10% fetal calf serum (FCS; Gibco-BRL) and 2 mML-glutamine (Invitrogen) (Complete RPMI), at 37° C. in 5% CO₂. AdheredRAW264.7 cells were infected with M. tubercluosis at a multiplicity ofinfection of 1:1. Four hours post-infection, macrophage monolayers werewashed with phosphate buffered saline (PBS), cells were incubated for anadditional 48 hours in fresh medium and total RNA was extracted for RTPCR analysis.

M. tubercluosis pellets from broth culture or M. tubercluosis-infectedmacrophages were resuspended in TRI reagent (Invitrogen) and disruptedwith 0.1-mm zirconia/silica beads in a BioSpec Products Bead Beater. RNAwas extracted, treated with TURBO DNase (Ambion) and resuspended inDPEC-treated water (Invitrogen) as described previously ((Muttucumaru,2004 #34). cDNA was synthesised from 1 μg of total RNA by usingSuperscript III reverse transcriptase (Invitrogen). Quantitive RT-PCRwas performed using 4 μL of cDNA, SYBR green I PCR Master Mix (Qiagen)and 5 μM of the gene-specific primer pair (supplementary table 1) in areaction volume of 25 μL. PCR reactions were run on a Rotogene6000-series sequence detector (Corbett research) in triplicate perprimer pair. Relative expression levels were determined using thecomparative threshold cycle method of Livak and Schmittgen ((Livak, 2001#99) using the non-induced M. tubercluosis 16S rRNA (encoded by rrs) asBthe control ((Banaiee, 2006 #101).

Human Studies

Subjects: 15 M. tubercluosis-infected, HIV negative patients wererecruited from the TB clinic at Royal Prince Alfred Hospital, MissendenRd, NSW, Australia. Peripheral blood mononuclear cells (PBMCs) wereobtained from biopsy or culture-proven patients of varying ages andgender that had or had not yet started anti-TB treatment. Ethicalapproval for this study was given by Sydney South West Area HealthService (protocol number: X06-0248). Patients were compared with 11healthy tuberculin skin test negative (TST-ve) individuals.

T cell proliferation assay: PBMCs from whole blood were isolated on aFicoll gradient (Histopaque-1077, Sigma Aldrich). 2.5×10⁵ cells/well ofPBMCs were incubated at 37° C. in 5% CO₂ for 5 days in the presence of10 μg/mL of the SAP proteins, 10 μg/mL of CFP, Ag-85B or 3 μg/mL ofConA. T cell proliferation was assayed by ³H-thymidine incorporation (MPBiomedicals, 1 μCi/well) at day 5 using liquid scintillationspectroscopy (Microbeta Luminescence Counter, Wallace). The lymphocytestimulation index (SI) was calculated using the following formula:average counts per minute (cpm) in the presence of antigen/average cpmin the absence of antigen. A SI of greater than or equal to 3 wasconsidered a positive response to antigen. Murine studies

Six- to eight-week-old female C57BL/6 mice were obtained from AnimalResources Centre (Perth, Australia) and maintained in specificpathogen-free conditions. For determination of immunogenicity, mice(4/group) were infected via the intra-nasal (i.n) route with 5×10⁴colony forming units (CFU) of M. tuberculosis Mt103. Three and 8 weekspost-infection single cell suspensions were prepared from themediastinal lymph node (MLN) of immunized mice in complete RPMI mediumand the number of interferon (IFN)-γ-producing cells was determined byELISpot as described previously ((Palendira, 2002 #210) using SAPenzymes, CFP and Ag85B at a concentration of 10 μg/ml with ConA used at3 μg/ml. For the analysis of protective efficacy, mice (5/group) wereimmunised subcutaneously (s.c) once with either 5×10⁵ CFU of M. bovisBCG, or 3 times at 2 week intervals with 10 μg of CysDNC proteinco-administered with dimethyl dioctadecyl ammoniumbromide (DDA) (1.25mg/ml) and monophosphoryl lipid A (MPL) (125 μg/ml) or intramuscularly(i.m) with 100 μg DNA vaccine per injection. Eight weeks after the finalvaccination, mice were challenged with aerosol M. tuberculosis Mt 103using an inhalation exposure apparatus (Glas-Col) with an infective doseof □100 viable bacilli per lung. Bacterial load was determined 4 weeksafter challenge by plating homogenates of lung and spleen.

Statistical Analysis

For assessment of protective efficacy, the significance of differenceswas evaluated by one-way ANOVA with pair-wise comparison ofmulti-grouped data sets achieved using the Bonferroni post hoc test. Forassessment of induction of host immune responses by SAP enzymes ofinfected mice or M. tuberculosis infected individuals compared touninfected mice or TST-ve individuals respectively, the Mann-Whitney's Utest was used (*P<0.05).

Results

Induction of M. Tubercluosis ATP Sulfurylase mRNA in the IntracellularEnvironment Correlates with Potent Antigen-Specific Immunity.

The sulphate activating complex (SAC) of M. tuberculosis is the firststep in the SAP (Supplementary FIG. 1) and constitutes 3 catalyticactivities, ATP sulfurylase, GTPase and APS kinase activity encoded bythe CysDNC operon (FIG. 1A). The ability of M. tubercluosis SAC toup-regulate its expression in culture conditions that mimicintracellular stress ((Pinto, 2004 #14), suggests that its expressionmay also be induced in the intracellular environment. To test this, weexamined the changes in CysDNC mRNA levels within RAW264.7 cells duringM. tuberculosis infection. We found that expression of CysDNC wassignificantly enhanced, displaying an approximately 4.4-fold increaseover the level found in broth-cultured bacilli during the first 48 hours(FIG. 1B). We also determined if expression of CysDNC was induced innon-replicating bacteria in order to mimic conditions encountered duringlatent infection. Using the nitric oxide donor DETA-NO ((Bryk, 2008#156) we were able to inhibit M. tubercluosis replication in vitrocompared to non-treated bacteria (FIG. 1C). M. tuberculosis CysDNC washighly up-regulated under conditions of non-replicating persistence anddemonstrated a 35-fold increase in gene expression compared to activelygrowing mycobacteria (FIG. 1D). This implies that CysDNC may be involvedin the ability of M. tubercluosis to adapt to the variety of stressesencountered in the intracellular environment and progression to thelatent state.

As CysDNC was induced at high levels within the intracellularenvironment and in non-replicating bacteria, we hypothesized that theenzyme may be recognized by the immune response during M. tuberculosisinfection. To test this, we intranasally infected mice with M.tubercluosis and examined the frequency of IFN-γ secreting cells in themediasteinal lymph nodes (MLN). At three weeks post infection,stimulation of MLN cells with CysDNC ex vivo resulted in a stronginduction of IFN-γ secreting T cells, which was similar to levelsinduced by the immuno-dominant secreted Ag85B protein of M. tubercluosis(FIG. 2A). This strong T cell response was maintained up to eight weekspost infection (FIG. 2B). Similar patterns of antigen-specific IFN-γsecreting cellular responses in response to CysDNC and Ag85B wereobserved in the lung (data not shown). In addition, lymphocyteproliferation assays of human PBMCs revealed that CysDNC was recognizedduring human M. tubercluosis infection (FIG. 2C). CysDNC responses weresimilar to that upon recall to Ag85B yet lower than that induced by CFP.However CysDNC significantly induced proliferation of PBMCs from TBpatients compared to TST-ve individuals (FIG. 2C). These resultsindicate that M. tubercluosis CysDNC, encoding ATP sulfurylase is apotent immuno-stimulatory antigen of M. tubercluosis.

Protective Immunity Against Virulent M. Tubercluosis Challenge FollowingVaccination with DNA Encoding ATP Sulfurylase.

The enhanced expression of the genes encoding ATP sulfurylase in theintracellular environment (FIG. 1B), in our non-replicating persistencemodel (FIG. 1C) and the ability of this protein complex to induce arobust antigen specific Th1 type cytokine response ex vivo (FIG. 2) mayrender the encoded products effective targets for anti-mycobaterialprotective immunity. To determine this, mice were immunised with DNAvectors expressing cysD and/or cysNC and aerosol challenged with M.tuberculosis. Immunisation with all vectors expressing either cysD orcysNC resulted in significantly reduced bacterial load compared to micevaccinated with the control vector, in both the lung (FIG. 3A) andspleen (FIG. 3B) (p<0.01). In all experiments, there was an increasingtrend for DNA-cysD to afford better protective efficacy than DNA-cysNCin the lung, while using a combination of DNA-cysD and DNA-cysNCequalled the protection seen with DNA-cysD alone. The protectiveefficacy was significantly greater in the spleen when mice wereimmunised with a combination of these two plasmids, and this protectionapproached the level achieved with BCG (FIG. 3B). Therefore ATPsulfurylase is a highly protective component of M. tuberculosis.

Downstream Enzymes of M. Tubercluosis Sulphate Assimilation Pathway(SAP) are Immunogenic Components of M. Tubercluosis.

The promising results obtained with M. tubercluosis ATP sulfurylase(CysDNC) led us to question whether other members of the SAP are targetsof the host immune response. We found that all SAP proteins tested weresignificantly up-regulated in the intracellular environment, with CysK1mRNA displaying the highest induction of approximately 6.7 fold (FIG.4A). The level of induction was similar for SirA and CysH while CysK1and CysE also displayed similar levels of intracellular up-regulation.This is in agreement with the fact the SirA and CysH are located withinthe same operon in the M. tuberculosis genome ((Cole, 1998 #225) whileCysK1 and. CysE lie adjacent in the genome ((Cole, 1998 #225;Schnell,2007 #87). We also determined that these proteins are recognized in M.tuberculosis infected mice, as all proteins induced IFN-γ secreting Tcells at 8 weeks post-infection (FIG. 4B). Like CysDNC (FIG. 2C),lymphocyte proliferation assays on human PBMCs revealed that all SAPenzymes studied were recognized during human M. tubercluosis infection(FIG. 4C).

As expression of all SAP enzymes was up-regulated in vivo and theproteins were recognized in mice and humans, we assessed if they couldimprove the protective efficacy afforded by DNA-CysDNC. When mice werevaccinated with DNA-CysDNC together with DNA encoding cysH, sirA, cysK1and cysE, we did not observe increases in protective efficacy comparedto DNA-CysDNC: alone both in the lung (FIG. 5A) or spleen (FIG. 5B).Therefore while all SAP members were recognized by the immune responsein M. tubercluosis infected humans and mice, CysDNC alone affordedmaximal protective efficacy in the mouse model used here.

Boosting BCG Vaccinated Mice with ATP Sulfurylase Improves ProtectionAfforded by BCG in the Lung Against Challenge with M. Tubercluosis.

Considering the strong recognition of C ATP sulfurylase by TB patientsand its protective effect in mice, we determined if this protein complexmay be a suitable candidate to boost the protective effect of BCG uponM. tuberculosis challenge. After low dose, aerosol delivery of M.tubercluosis, naive mice demonstrated substantial bacterial growth inthe lungs and dissemination to spleens were detected (FIGS. 6A and 6B).In contrast, immunisation with BCG alone resulted in significantprotection against M. tuberculosis challenge with an approximate1.5-log₁₀ reduction in the M. tubercluosis load in the lung and spleen(FIGS. 6A, 6B). Boosting with CysDNC protein led to a furthersignificant reduction compared to vaccination with BCG alone of 0.5-log10 M. tubercluosis in the lung (FIG. 6A). While bacterial burden wasreduced in the spleen with boosting, this difference did not achievesignificance (FIG. 6B). Therefore CysDNC is able to improve theprotective effect of the BCG against M. tuberculosis infection, whichwas most apparent in the lung.

Discussion

The identification of new targets of host immunity would markedly aidefforts to develop more effective TB vaccines. In this report weidentify the sulphate activation complex (SAC) of M. tuberculosis as amajor antigenic component of the bacillus. The SAC is an enzyme complexwith 3 catalytic activities ((Pinto, 2004 #14;Sun, 2005 #13). Thiscomplex is predicted to play a role in adaption of M. tuberculosis tothe host cell environment, due to up-regulation of CysDNC withinmacrophages (FIG. 1B) ((Schnappinger, 2003 #31), and in response to anumber of in vitro stress conditions including nutrient starvation andoxidative stress ((Hatzios, 2011 #167;Pinto, 2004 #14). Therefore it ispossible that strong recognition of CysDNC by both TB patients (FIG. 2C)and M. tuberculosis-infected mice (FIGS. 2A&B) may be due to theenhanced expression of CysDNC within the host. Intriguingly, CysDNC alsodisplayed significant up-regulation in a model of non-replicating growthof M. tubercluosis (FIG. 1D). This result implies that CysDNC-expressionmay be required for the adaption of M. tubercluosis to the latent stateduring chronic infection. This is supported by the role of reducedsulphur compounds in the onset of chronic M. tubercluosis infection((Senaratne, 2006 #3) and corresponds to the sustained recognition ofCysDNC by T cells at late-stages of M. tubercluosis infection in mice(FIG. 2B). This further implies that M. tuberculosis CysDNC maycontribute to the organism's survival in a state of non-replicatingpersistence by sustaining metabolic pathways that utilize cysteine.Cysteine is incorporated into Acetyl CoA, the building block for lipidsin the organism's cell wall and a substrate for the glyoxylate shunt, apathway required for M. tubercluosis to persist in macrophages and inmice ((McKinney, 2000 #211). Further, CysDNC is up-regulated duringlow-oxygen dormancy ((Voskuil, 2004 #35) and plays a role in thesuppression of apoptosis by macrophages ((Danelishvili, 2010 #116),implying a role for CysDNC in evasion of the host immune response.Interestingly cysD was found to be up-regulated after exposure toanti-TB drugs that are bactericidal to non-replicating bacteria ((Fu,2009 #117;Heifets, 2005 #120;Tasneen, 2008 #12 I).

Enhanced recognition of mycobacterial antigens by the host immuneresponse does not always correlate protection against challenge withvirulent M. tubercluosis in animal models ((Gartner, 2007 #215;Kamath,1999 #143;Skinner, 2003 #115). We therefore assessed if CysDNC wasprotective in our low-dose murine model of aerosol M. tubercluosisinfection. When delivered as DNA vaccines, CysD and CysNC wereprotective as single components in both the lung and spleen, and acombination of the two constructs achieved a level of protection similarto that induced by BCG (FIG. 3). Thus the strong expression of genesencoding CysDNC correlates with the protective effect of the antigeniccomplex in the models used here. However, addition of DNA vaccinesencoding other components of the SAP did not improve the protectiveeffect of CysDNC, despite the fact that proteins were recognised byPBMCs from TB patients, were up-regulated within macrophages and inducedstrong IFN-□ responses from T cells of M. tubercluosis-infected mice(FIG. 4). It is unclear as to why CysDNC appears to be dominant memberof the SAP in terms of protective efficacy, but may relate toMHC-restricted responses based on the mouse strain used in this study.It is also of interest to note that the SAP antigens are proposed to beintracellular or membrane-associated proteins due to their function insulfur metabolism ((de Souza, 2011 #206), and we have confirmed this forsome of the members by Western blot (not shown). However, all M.tubercluosis antigens in clinical trials are secreted proteins, as theseare predicted to be the early targets of host immunity ((Kaufmann, 2011#196). This study suggests that non-secreted proteins may also besuitable components of new TB vaccine formations. Further, the recentidentification of stress-induced, latency-associated antigens improvingprotection at late-stages post M. tuberculosis infection ((Aagaard, 2011#213) warrants further testing of CysDNC in similar models, consideringthe marked induction of the protein complex in non-replicating bacteriaand in response to in vitro stress.

An important property of potential subunit vaccines is the capacity to‘boost’ protection with prior BCG immunization, as this is the proposedrole for such vaccines in new TB vaccine schedules ((Kaufmann, 2011#196). A handful of proteins have demonstrated an ability to boostBCG-induced protection in experimental M. tuberculosis infection ((Lu,2011 #144;Dey, 2011 #145;Rouanet, 2009 #214). CysDNC can now be added tothis list, as the antigen complex was able to significantly increase theprotective effect of BCG alone in the lung, the primary site ofinfection in the model used (FIG. 6).

In addition to defining the antigenic role of the SAP proteins, thisstudy has also expanded our knowledge of regulation of SAP geneexpression during infection. All components of the SAP tested displayedup-regulation within the macrophage cell line used here. This adaptationto the phagosome environment is an indication of the bacterium's abilityto supply cysteine to the cell in a timely fashion, which has beenextensively reviewed by Hatzios and Bertozzi ((Hatzios, 2011 #167). Itmay be that enzymes in the SAP regulate the expression of each other.For instance serine acetyl transferase has been shown to associate andform a complex with the last enzyme in the pathway o-acetlyserinesulthydralase (OASS) ((Droux, 1998 #218). While this interactionnegatively regulates OASS ((Mino, 2000 #222;Schnell, 2007 #87), similarto ATP-sulfurylase, it's expression and activity may depend on therequirement for cysteine ((Kredich, 1966 #219;Mino, 1999 #220;Mino, 2000#221;Pinto, 2004 #14). Further more, E. coli ATP sulfurylase forms atight complex with, OASS ((Wei, 2002 #96) and it is this complex thatcan activate sulphate to produce APS (supplementary FIG. 1). It has beensuggested that given the similarities between E. coli and M.tubercluosis ATP sulfurylases, that the mycobacterial system also formsa higher order complex, linking catalytic functions with other enzymesin the SAP ((Sun, 2005 #13). This has not been formally tested, however,it may be the reason why all of the enzymes of the SAP induce robusthost immune responses (FIG. 2, FIGS. 4B &C). Also highlighting theregulation of sulphur metabolism genes is their enhanced transcriptionupon exposure to various antibiotics which may influence how M.tubercluosis responds to these drugs ((Hatzios, 2011 #167). Presumablydrug-responsive genes encode proteins that are relevant to the drugsmode of action, and many sulphur-metabolising enzymes are promising drugtargets ((Bhave, 2007 #1).

In summary, we have identified components of the SAP as host-cellinduced proteins that are major antigenic components of M. tubercluosis.

Example 2

The preferred niche within the host of virulent mycobacteria and theattenuated BCG vaccine strain is the intracellular environment of themacrophage. It has been observed that mycobacteria also have the abilityto infect and persist within DCs and stimulate in vivo T cell responses[8,9].

We hypothesized that the targeted expression of antigen by recombinant(r) BCG within DCs may influence the subsequent generation ofantigen-specific immunity. We considered that the promoter controllingexpression of the M. tuberculosis hspX gene may be a suitable candidateto direct expression of antigen to APCs after vaccination with BCG.

HspX, or α-crystallin protein, is a member of the small molecular weightheat-shock proteins that act as ATP-independent chaperones [10,11]. ThehspX promoter is rapidly induced upon entry of M. tuberculosis intocultured macrophages [12]. The gene is also up-regulated in vitro byhypoxia [12], during stationary phase of growth of aerated M.tuberculosis cultures [11] and by addition of NO donors [13], which mayall represent conditions encountered by M. tubercluosis within hostcells. Importantly, the hspX gene is up-regulated after infection ofmice with virulent M. tuberculosis [14].

In this report, we demonstrate that the hspX promoter can be used tomodulate expression of antigens expressed by rBCG, and antigenexpression is rapidly induced upon entry of rBCG within DCs. Use of thehspX promoter to control antigen expression resulted in acceleratedpriming of antigen-specific T cells and sustained in vivo generation ofantigen-reactive T cells after vaccination.

Materials and Methods

Bacterial Strains, Media and Antigens

M. tuberculosis 1437Rv (ATC27294) and M. bovis BCG (Pasteur strain) weregrown on Middlebrook 7H9 medium (Difco, BD) supplemented with 0.5%glycerol, 0.05% Tween-80 and 10% ADC or on solid Middlebrook 7H11 medium(Difco, BD) supplemented with OADC. When required, the antibiotickanamycin (Km) was added at a concentration of 20 μ/ml.

Mice

Six to eight weeks old C57BL/6 mice were obtained from. Animal ResourcesCentre (Perth, WA, Australia) and maintained in specific pathogen-freeconditions. The p25 CD4⁺ TCR transgenic (specific for residues 240-254of Ag85B) were obtained from Prof. K. Takatsu (University of Tokyo,Japan) and Prof. J. Ernst (New York University School of Medicine, NY)[15] and were backcrossed onto B6.SJL/Ptprca to obtain the p25⁺CD45.1⁺line. Animal experiments were performed with approval of the Universityof Sydney Animal Care and Ethics Committee.

Construction of Recombinant (r)BCG Strains

BCG expressing GFP under control of the M. tubercluosis hspX promoter(BCG:P_(hspX)-GFP) was constructed by transformation of plasmidpMV306:GFP into BCG, which was a kind gift of Professor Cliff Barry,Tuberculosis Research Section, NIAID, National Institutes of Health,Rockville, Md. To develop BCG in which the hspX promoter drivesexpression of the Ag85B protein, the Ag85B protein-encoding fbpB genewas amplified from M. tuberculosis genomic DNA and used to replace thegfp gene in pMV306:GFP, resulting in the intermediate vector pJEX88. TheP_(hspX)-fbpB fragment was excised by digestion with XbaI and HpaI andligated to the shuttle-vector pMV261 [16] digested with the sameenzymes. The resultant plasmid was transformed into BCG to produceBCG:P_(hspX)-85B. Control BCG used in this study was either BCG Pasteurstrain or BCG Pasteur transformed with pMV261 [16]. Construction of rBCGover-expressing the M. tubercluosis Ag85B protein under the control ofthe hsp60 promoter (BCG:P_(hsp60)-85B) has been described previously[17].

Dendritic Cell Infections

DCs were prepared form the bone marrow of mice as described previously[18]. For DC infection, 4-day old DC cultures were incubated with rBCGstrains at a multiplicity of infection of 5:1 or 1:1. After 4 h,extracellular bacteria were removed by extensive washing and at 6 and 24h post-infection, DCs were lysed and bacteria collected for flowcytometry (LSR-II, Becton Dickinson, San Jose, Calif., USA). The foldincrease in fluorescence was determined by dividing the fluorescence ofbacteria populations at 6 and 24 h by the initial value (day 0). Forvisualisation by confocal microscopy, similar infection conditions wereused, except DCs were allowed to adhere onto round 25 mm coverslips(Leica Microsystems, Wetzlar, Germany) prior to rBCG infection and thenviewed under the LP5 Confocal microscope (Leica).

In Vitro Immunogenicity Assays

Four-day DC culture (1×105 cells) were infected with Rbcg strains at anMOI of 5:1. CD4 T cells were purified from p25⁺CD45.1⁺ mice by autoMACSseparation (Miltenyi Biotec, Bergisch Gladbach, Germany) and 5×105purified T cells cultured with infected DCs. Three days followingco-incubation, supernatant were assayed for IFN-γ by ELISA as describedpreviously [19] and T cell proliferation was measured by [3H]-thymidineuptake.

In Vivo Immune Response

To detect GFP expression in vivo, C57BL/6 mice were inoculated with1×107 CFUs of fluorescent rBCG strains subcutaneously in the footpads.Tissue of the local inflammatory site was obtained immediately afterinfection or at days 1, 3 or 7 post-infection. Tissues were digestedwith collagenase and DNAse for at least 1 h and then strained to recovercells. Cells were stained with CD45-APCCy7, CD11b-APC and CD11c-PE-Cy7(BD Pharmingen). Stained cells were analysed using the LSR-II flowcytometer (BD).

For determination of T cell priming after rBCG delivery, lymph nodecells from p25⁺CD45.1⁺ mice were prepared and labelled withCarboxyfluorescein succinimidyl ester (CFSE; Molecular Probes,Invitrogen, USA). C57BL16 mice received 5×105 CFSE-labelled p25 lymphnode cells i.v. and the next day were immunized subcutaneously with5×105 CFU rBCG strains. At 3 or 7 days postvaccination organs wereprocessed and the CFSE profile of dividing cells analysed.

For determination of rBCG immunogenicity, C57BL/6 mice weresubcutaneously vaccinated with 5×105 CFUs of rBCG strains. At 3 and 12weeks post-vaccination, splenocytes were recovered and 2×105 splenocyteswere cultured with p25 peptide (3 μg/ml) at 37° C. in 5% CO₂. Eighteenhours following co-incubation, the number of IFN-γ secreting cells wasdetermined by ELIspot as described previously [19] and at 72 h followingthe antigen challenge, T cell proliferation was assessed by[3H]-thymidine uptake.

Protective Efficacy

For assessment of protective efficacy, C57BL/6 mice (5 per group) wereimmunised with 5×105 CFU of BCG strains and at 12 weeks post-vaccinationmice were challenged with aerosol M. tuberculosis H37Rv using aMiddlebrook airborne infection apparatus (Glas-Col, Terre Haute, Ind.,USA) with an infective dose of approximately 100 viable bacilli perlung. Four weeks after the challenge the number of bacteria within thelung and spleen was enumerated on Middlebrook 7H11 Bacto agar.

Statistical Analysis

The significance of differences for linear and log-transformed assayswas determined by analysis of variance (ANOVA) using Bonferroni'sMultiple Comparison test was used for pair-wise comparison ofmulti-grouped data sets. Differences with p<0.05 were consideredsignificant.

Results

Induction of the M. Tuberculosis hspX Promoter within Dendritic Cells

We hypothesized that targeting antigen expression to DCs duringmycobacterial infection would have a positive effect on the resultantimmune response directed towards the antigen. In order to induceexpression of foreign genes within DCs, we constructed BCG strains inwhich the M. tubercluosis hspX promoter (P_(hspX)) controlled geneexpression. BCG:P_(hspX)-GFP, in which gfp was expressed using P_(hspX),displayed marked up-regulation of GFP fluorescence only when grown innon-aerated cultures, confirming up-regulation of the hspX promoterunder low oxygen tension (FIG. 7A). After infection of DCs for 24 h withBCG:P_(hspX)-GFP, isolated bacteria exhibited a dramatic increase influorescence when compared to the initial inoculum (day 0) or bacteriaisolated 6 h post-infection (FIG. 7B). Visualisation of GFP fluorescenceby confocal microscopy confirmed the induction of hspX-controlledexpression of GFP within rBCG-infected DCs (FIG. 7C). These resultsdemonstrate that the hspX promoter can be used to drive rapid andpronounced induction of gene expression by recombinant BCG within DCs.

Early In Vivo Induction of hspX-Induced Expression after Vaccination

In order to determine if the hspX promoter could be used to driveantigen expression in vivo, mice were vaccinated with BCG:P_(hspX)GFPand the presence of GFP host cells determined. GFP⁺ cells were detectedas early as 1 day post-vaccination, and were prominent at days 3 and 7post-vaccination (FIG. 8A). The proportion of GFP⁺ cells peaked at day 3post-vaccination, however there were no significant differences betweenthe three timepoints examined (FIG. 8B). We also detected an influx ofCD11c^(hi)CD11b^(hi) DCs to the site of rBCG vaccination, with numberspeaking at day 7 postvaccination (FIG. 9A). GFP⁺ DCs were prominent atlater stages of infection, with a significant number of DCs harbouringBCG:P_(hspX)GFP in an induced state at day 3 and day 7 respectively(FIGS. 9B and C). These data indicate that vaccination withBCG:P_(hspX)-GFP results in infection of DCs and rapid intracellularinduction of the hspX promoter.

Use of the hspX Promoter to Drive Expression of Foreign Antigens in BCG

To determine if the hspX promoter could be used to modulate immunity todefined antigens, we expressed the gene encoding the immunodominant M.tubercluosis Ag85B antigen under the control of this promoter. Althougha homologue of this protein is expressed by BCG, improved anti-Ag85Bimmunity is conferred on BCG by constitutive over-expression of theantigen [17.] The BCG:P_(hspX)-85B strain displayed up-regulation ofAg85B expression in cultures grown under conditions of limited aeration(data not shown). To determine if hspX-mediated expression influencespresentation of Ag85B by DCs, in vitro cultured DCs from mouse bonemarrow were infected with BCG:P_(hspX)-85B, BCG alone orBCG:P_(hsp60)-85B, in which Ag85B is constitutively expressed under thecontrol of the strong hsp60 promoter [17]. Infected cells were culturedwith transgenic Ag85B-specific CD4⁺ T cells (p25 T cells [15]) andproliferation and cytokines release examined. DCs infected with all 3strains induced p25 T cell proliferation (FIG. 10A) and IFN-γ secretion(FIG. 10B), which was not observed in uninfected cells. Responses weremore prominent in DCs infected with BCG:P_(hspX)-85B, and thisdifference reached significance in T cell proliferation assays (FIG.10A). Therefore the hspX promoter can be used to drive foreign antigenexpression in BCG, and induction within DCs results in improved T cellproliferation in vitro.

Augmented In Vivo T Cell Immunity Induced by Vaccination withBCG:P_(hspX)-85B

The in vitro immunogenicity experiments suggested that use of the hspXpromoter might be able to modify BCG-induced immunity in vivo.Considering the rapid induction of expression driven by the hspXpromoter after vaccination (FIG. 8), we first determined if vaccinationwith BCG:P_(hspX)-85B led to improved early priming of Ag85B reactive Tcells compared to BCG alone or BCG:P_(hsp60)-85B. To do this,CFSE-labelled p25 T cells were transferred into wild-type recipients,and the activation and proliferation of the transferred cells examinedafter vaccination with BCG strains. At day 3 post-infection,BCG:P_(hspX)-85B vaccination had induced p25 T cells to proliferate,with most divided cells displaying an CFSE intermediate or low profile,while vaccination with BCG alone or BCG:Phsp60-85B did not result inappreciable proliferation (FIGS. 11A and B). At day 7 post-infection allBCG strains induced proliferation of the majority of transferred p25 Tcells as determined by the CSFE profile of p25 CD4⁺ T cells (FIG. 11A)with approximately 90% of p25 CD4 T cells displaying a CSFE low profileafter vaccination with BCG, BCG:Phsp60-85B or BCG:P_(hspX)-85B (FIG.11B). Taken together, these results indicate that induction of Ag85B inBCG:P_(hspX)-85B accelerates the initial priming of p25 T cells, howeverboth BCG and BCG:P_(hsp60)-85B produced sufficient antigen to result inproliferation of p25 T cells at later timepoints.

We next assessed the long-term T cell responses by vaccinating mice withthe three BCG strains and examining the generation of Ag85B-sepecific Tcells at either 3 or 12 weeks post-vaccination. At 3 weekspost-vaccination, both BCG:P_(hsp60)-85B and BCG:P_(hspX)-85B led to theincreased generation of Ag85B-specific IFN-γ-secreting cells compared toBCG alone, with BCG:P_(hspX)-85B in particular increasing the number ofresponding cells by approximately 5-fold compared to control BCG (FIG.12A). At 12 weeks post-vaccination both BCG and BCG:P_(hsp60)-85Bdisplayed equivalent levels of IFN-γ-secreting cells responding toAg85B, which were significantly greater than that observed inunvaccinated mice (FIG. 12B). However, in mice vaccinated withBCG:P_(hspX)-85B the number of antigen specific IFN-γ-secreting cellswas approximately 6 times greater than that induced by BCG alone andmore than 3 times the response observed after vaccination withBCG:Phsp60-85B (FIG. 12B). These results indicate that the ability toinduce Ag85B expression within host cells results in a pattern ofincreased in vivo T cell immunity, which is most evident at extendedtimepoints postvaccination.

Since Ag85B is an immunodominant antigen of M. tuberculosis, wedetermined if rBCG expressing Ag85B under control of the hspX promotercan improve the protective effect of BCG against M. tuberculosisinfection. Twelve weeks post-vaccination, mice were challenged with lowdose M. tuberculosis strain H37Rv via the aerosol route. The bacterialload in both the lungs (FIG. 12C) and the spleen (FIG. 12D) at 4 weekspost M. tubercluosis infection were significantly reduced in all micevaccinated with BCG strains compared to unvaccinated mice. However, theprotective effect was similar in mice vaccinated with BCG alone,BCG:P_(hspX)-85B, BCG:P_(hsp60)-85B, indicating that in this model Ag85Boverexpression did not improve protective efficacy.

Discussion

The BCG vaccine displays variable protective efficacy againsttuberculosis, however the vaccine can be engineered to express foreignmolecules in a functional form, and this has driven the development ofBCG as a recombinant vector to protect against infectious diseases andmalignancies such as cancer [20]. Critical to the success of suchapproaches is the ability to modulate BCG-induced immunity to generatethe desired immune response. In this report, we show that the M.tuberculosis hspX promoter can be used to modulate expression ofrecombinant antigens produced in BCG. We demonstrate that the hspXpromoter is rapidly induced in vitro within DCs (FIG. 6), whichcomplements existing literature on hspX expression within macrophages[21] and the uptake of BCG by DCs after infection of mice [8]. Thisrapid and pronounced in vivo induction of the hspX promoter within DCsis important for the use of this antigen carrier system due to thecritical role of DCs in the initiation of mycobacterial immune responses[8,22,23]. We also demonstrate that hspX promoter activity is evidentwithin DCs from rBCG-vaccinated mice as early as 24 h post-infection(FIG. 8). This rapid promoter induction appears to contradict with hspXtranscript levels after low-dose aerosol M. tuberculosis infection ofmice, with hspX mRNA detected only at approximately 15 dayspost-infection and most prevalent at later stages [14]. This suggeststhat in vivo hspX accumulation in M. tuberculosis may be a slow process,or could also relate to the delivery of a high rBCG dose used in thecurrent study and the sensitive GFP reporting system facilitating thedetection of hspX promoter activity.

In order to determine the antigen-specific effects of P_(hspX)-drivenexpression, we made use of the hspX promoter to modulate expression ofM. tuberculosis Ag85B, a secreted mycobacterial protein that is acomponent of a number of candidate tuberculosis vaccines [24]. Weobserved that the early recruitment of DCs to the infection site andrapid phagocytosis of BCG (FIG. 9) correlated with enhanced priming andactivation of Ag85B-reactive T cells after vaccination withBCG:P_(hspX)-85B (FIGS. 10 and 11). Further, this improved T cellpriming resulted in sustained generation of IFN-γ-secreting cellsrecognising Ag85B up to 3 months postvaccination. Intriguingly,expression of the gene encoding Ag85B is down-regulated during chronicM. tuberculosis infection in mice [14], and it would be of interest todetermine if vaccines which use the native Ag85B promoter to driveantigen expression in BCG can sustain long-term protective immunity inhumans [25]. We also used a murine model of aerosol M. tuberculosisinfection to determine if in vivo induction of Ag85B could improve theprotective effect of the BCG vaccine. However, we did not observe animproved protective effect compared to BCG alone (FIG. 12). In aprevious study, we similarly observed that overexpression of

Ag85B in BCG does not improved the protective effect of BCG, despiteincreasing anti-Ag85B immunity in vaccinated mice [17]. This suggeststhat other antigens may need to be assessed in this system to determinethe effect of P_(hspX)-driven expression in BCG to protect against M.tubercluosis infection, or alternatively BCG:P_(hspX)-85B should beassessed in other pre-clinical models of tuberculosis where Ag85Bexpression doe impart some level of improved protective efficacy whenexpressed in rBCG [25].

The mechanism of improved and sustained immunity due to up-regulation ofantigen via the hspX promoter is unclear. Constitutive over-expressionof Ag85B using the mycobacterial hsp60 promoter leads to very strongexpression levels in rBCG as detected by western blotting [17] and issuperior to BCG alone at stimulating antigen specific T cell immunityearly after vaccination, however this effect is not sustained long-term(FIGS. 12A and 12B). It is possible that P_(hsp60) transcription isdown-regulated at later stages of BCG infection, thus resulting inreduced antigen levels at extended timepoints, or instability of thepromoter in vivo reduces immunogenicity [26]. Conversely, P_(hspX)activity appears to be maximal at longer timepoints post-infection [14],indicating that this approach may result in heightened and sustainedlevels of antigen expression by BCG:P_(hspX)-85B. It has been previouslyreported that Ag85B over-expression in BCG leads to improved autophagyand,improved capacity of the rBCG strain to stimulate antigen-specific Tcell responses [27]. When we inhibited autophagy in rBCG-infected. DCsusing 3-methyladenine and determined the extent of activation of Ag85Bspecific T cells, we observed a trend of reduced T cell activation intreated DCs infected with BCG:P_(hspX)-85B, however this effect did notreach statistical significance in all experiments (data not shown).

The results presented in the current study demonstrate that the capacityto induce antigen expression using rBCG as the carrier leads tosignificant and sustained generation of cellular immune responses. Thishas clear implications for diseases that require ‘Th1’ like responsesfor their control, such as tuberculosis.

Example 3

Methods

C57BL/6 mice (n=5) were immunized 3 times by s.c injection with eitheradjuvant (MPL/DDA), Ag85B-CysD fusion protein (10 mg) or Ag85B (10 mg).At the time of the first injection of protein vaccines, mice wereimmunized once by s.c injection with 5×10⁵ CFU of BCG. Four weeksfollowing the third immunization, mice were challenged with aerosol M.tubercluosis with an infective dose of ˜100 viable bacilli per lung.

Results

Bacterial load was determined in the lung (FIG. 14A) and the spleen(FIG. 14B) 4 weeks after immunised mice were challenged with aerosol M.tubercluosis. Data are shown as the mean CFU (±SEM) per organ. Thesignificance of the differences between groups was evaluated by one-wayANOVA with pair-wise comparison of multi-grouped data sets achievedusing the Bonferroni post hoc test.

Vaccination with Ag85B-CysD resulted in protection equivalent to theexisting BCG vaccine. This is a remarkable result considering BCGexpresses a large number of antigenic targets, yet our fusion of twoantigens is equally protective (FIG. 14).

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TABLE 1 SupplementaryPrimer and clones for protein purification used in this studyFor Real-time PCR Gene annotation Gene name Forward primerReverse primer cysDN ATP sulfurylase 5′-agt cat cgc cga aac5′-ttg cca tcg tcg acg tgc-3′ gaa-3′ cysH APS reductase5′-gac atc gcg ggt gga 5′-gac gga etc gat cgc atc ca-3′ tc-3′ sirASulphite reductase 5′-gtt cga gca cag cat 5′-gtc cgt cgt cga tca tctttg gt-3′ gt-3′ cysK1 O-Acetyl-serine Sulfhydralse5′-ttc tcg aac cca cga 5′-ccc gga gtg agg atg agt gcg-3′ tc-3′ cysESerine Acetlysulfhydralse 5′-ttc atc gac cac gcg5′-gat cgg acc gag gac ctt acc-3′ g-3′ rrs 16s rRNA5′-agg cag cag tgg gga 5′-cta ccg tca atc cga gag ata-3′ aa-3′For DNA vaccine cloning-all genes cloned into pcDNA3.Restriction Enzyme site used in cloning is highlighted. Forward primerReverse primer DNA-cysDcysD was cut out of a pET22b (Novagen) vector using BamHI and XhoI restrictionenzymes and cloned into pcDNA3 cut with the same enzymes DNA-cysNcysN was cut out of a pET22b vector using BamHI and NotI restriction enzymesand cloned into pcDNA3 cut with the same enzymes DNA-cysH5′-acg aaa gct tat gag cgg cga gac a-3′5′-ggg ggg atc ccg agg cgt gca acc c-3′ DNA-sirA5′-tag gaa gct tat gtc cgc gaa gga g-3′5′-tcg cgg tac ctc gca ggt cgt cct c-3′ DNA-cysK15′-ggc gaa gct tat gag cat cgc cga g-3′5′-gca tgg atc cgt cagcca cgt cgg c-3′ DNA-cysE5′-tga caa gct tat gct gac ggc cat g-3′5′-tat tgg tac cga tcg aga agt cct c-3′ For protein purificationDescription Reference CysDN Described previously((Pinto, 2004; Sun, 2005) CysH Described previously ((Sun, 2006) SirADescribed previously ((Pinto, 2007) CysK1Cloned into pET-23A (Noavagen) using ((Hara, 1990; Wei, 2002)Nde I and BamH I restriction enzymesand purified as described previously CysECloned into a dual-tag expression ((Sun, 2005)vector derived from pGEX vector ((Andreassi, 2004 #98) using Nde I andNot I restriction enzymes and was purified as described previously Ag85BCloned into pET19b (Noavagen) expression vector with NdeI & BamHIrestriction enzymes and purification of this protein was carried outthrough standard His-tag purification protocols (Qiagen)

(CysD nucleic sequence) SEQ ID No: 1ATGGCAATAACCATAAATATGGTCAATCCTACCGGATTTATCAGGTATGAGGACGTGGAACAGGAAGCCATGACCAGCGATGTGACGGTGGGCCCCGCACCCGGCCAGTACCAACTGAGCCATCTGCGCTTGCTGGAGGCCGAAGCCATCCACGTCATCCGGGAGGTGGCCGCCGAGTTCGAGCGGCCAGTGCTGTTGTTCTCGGGGGGCAAGGACTCCATCGTCATGCTGCACCTGGCGCTGAAGGCGTTTCGGCCCGGGCGACTGCCGTTCCCGGTCATGCACGTCGACACCGGTCACAACTTCGACGAAGTTATCGCTACCCGAGACGAGTTGGTCGCCGCGGCCGGGGTGCGGCTGGTGGTGGCGTCGGTGCAGGACGATATCGATGCCGGTCGGGTCGTCGAGACCATCCCGTCGCGAAATCCGATACAGACCGTGACGCTGCTGCGGGCCATCCGGGAGAACCAATTCGACGCGGCATTCGGGGGAGCCCGGCGCGACGAGGAGAAGGCCCGCGCCAAGGAGCGGGTGTTCAGCTTCCGCGACGAGTTCGGCCAGTGGGACCCGAAGGCTCAGCGGCCGGAACTGTGGAACCTCTACAACGGACGGCACCACAAGGGCGAGCACATCCGGGTCTTCCCGCTGTCCAACTGGACCGAATTCGACATCTGGTCCTACATCGGCGCCGAGCAGGTCAGGCTGCCGTCCATCTATTTCGCCCACCGGCGCAAGGTGTTTCAGCGCGACGGCATGTTGCTGGCCGTGCACCGGCACATGCAACCGCGAGCCGACGAGCCGGTGTTCGAGGCCACGGTGCGATTCCGCACCGTCGGGGATGTTACCTGCACCGGGTGCGTCGAGTCGTCGGCATCGACGGTCGCGGAAGTCATCGCCGAAACTGCGGTGGCCCGCTTGACGGAGCGCGGGGCGACCAGGGCTGACGACCGGATCTCGGAGGCTGGAATGGAAGACCGCAAGCGGCAGGGATACTTCTGA(CysD amino acid sequence) SEQ ID No: 2MAITINMVNPTGFIRYEDVEQEAMTSDVTVGPAPGQYQLSHLRLLEAEAIHVIREVAAEFERPVLLFSGGKDSIVMLHLALKAFRPGRLPFPVMHVDTGHNFDEVIATRDELVAAAGVRLVVASVQDDIDAGRVVETIPSRNPIQTVTLLRAIRENQFDAAFGGARRDEEKARAKERVFSFRDEFGQWDPKAQRPELWNLYNGRHHKGEHIRVFPLSNWTEFDIWSYIGAEQVRLPSIYFAHRRKVFQRDGMLLAVHRHMQPRADEPVFEATVRFRTVGDVTCTGCVESSASTVAEVIAETAVARLTERGATRADDRISEAGMEDRKRQGYF (CysNC DNA sequence) SEQ ID No: 3ATGACGACGCTATTGCGGCTGGCGACAGCGGGTTCCGTCGACGATGGCAAGTCCACGCTGATTGGGCGGCTACTCTACGACTCCAAGGCTGTGATGGAAGACCAGTGGGCGTCGGTGGAGCAAACGTCCAAGGACCGGGGCCACGACTACACCGACCTGGCTCTGGTCACCGACGGCCTGCGGGCCGAGCGGGAACAGGGCATCACCATCGACGTTGCCTACCGCTACTTCGCCACTCCCAAGCGGAAATTCATCATTGCCGACACCCCGGGACACATCCAATACACCCGCAACATGGTGACCGGTGCGTCCACCGCCCAACTGGTGATCGTACTGGTGGATGCCCGGCACGGCTTGCTGGAGCAATCCCGCCGGCACGCCTTCCTGGCGTCGCTGCTGGGCATCCGCCACCTGGTGCTCGCGGTCAACAAGATGGACTTGCTTGGCTGGGACCAAGAGAAATTCGACGCGATTCGAGACGAATTCCACGCCTTCGCGGCCCGCCTCGACGTGCAGGACGTCACCTCCATCCCAATCTCCGCGCTGCACGGCGACAACGTGGTGACCAAATCCGACCAGACGCCCTGGTACGAGGGACCGTCGCTGCTGTCGCATCTCGAAGACGTCTACATCGCCGGTGACCGCAACATGGTCGACGTGCGATTCCCGGTCCAGTACGTCATCCGGCCGCACACCCTCGAGCATCAAGACCACCGCAGCTACGCGGGCACCGTGGCCAGTGGGGTAATGCGTTCAGGCGACGAAGTTGTCGTGCTGCCGATCGGTAAGACCACCCGGATCACCGCGATCGACGGCCCGAACGGCCCGGTGGCAGAAGCGTTTCCGCCGATGGCGGTTTCGGTGCGGCTCGCCGACGACATCGATATCTCGCGTGGTGACATGATCGCTCGCACCCACAACCAGCCCAGGATCACACAAGAATTCGACGCGACCGTGTGCTGGATGGCCGACAACGCGGTGCTAGAGCCCGGCCGCGACTACGTTGTCAAGCACACCACCCGAACCGTCCGCGCGAGGATAGCCGGGCTGGATTACCGGCTCGATGTCAACACCCTGCATCGCGACAAGACCGCAACGGCGTTGAAACTCAACGAACTGGGCCGTGTTTCGCTGCGCACCCAGGTGCCGTTGCTGCTTGACGAGTACACCCGCAACGCTAGCACCGGCTCGTTCATCCTCATTGACCCCGACACCAACGGAACGGTGGCGGCGGGCATGGTGTTACGCGACGTCTCGGCCCGCACGCCTAGCCCGAACACGGTGCGGCACAGATCGCTCGTCACTGCGCAAGATCGGCCGCCCAGGGGCAAGACGGTGTGGTTTACCGGACTGTCCGGCTCCGGCAAGTCGTCGGTGGCCATGCTGGTTGAGCGGAAGCTACTCGAAAAGGGCATCTCCGCTTACGTTCTGGACGGCGACAACCTACGGCATGGCCTCAACGCCGACCTGGGCTTTTCCATGGCCGACCGCGCGGAGAACCTGCGCCGGCTGTCGCATGTGGCCACACTGCTCGCCGATTGTGGCCACCTGGTGCTGGTGCCCGCGATCAGCCCCCTTGCTGAGCACCGTGCCCTGGCTCGTAAAGTGCACGCTGATGCGGGAATCGACTTTTTCGAGGTGTTCTGTGACACCCCGCTGCAGGACTGTGAGAGGCGTGATCCCAAAGGGTTGTACGCCAAAGCGCGTGCGGGTGAGATCACGCACTTCACCGGGATCGACAGCCCATATCAGCGGCCCAAGAACCCAGACCTACGGCTTACGCCGGATCGCAGCATAGACGAGCAGGCGCAGGAGGTTATCGACCTGTTGGAGTCATCGTCTTAG (CysNC amino acid sequence) SEQ ID No: 4MTTLLRLATAGSVDDGKSTLIGRLLYDSKAVMEDQWASVEQTSKDRGHDYTDLALVTDGLRAREQGITIDVAYRYFATPKRKFIIADTPGHIQYTRNMVTGASTAQLVIVLVDARHGLLEQSRRHAFLASLLGIRHLVLAVNKMDLLGWDQEKFDAIRDEFHAFAARLDVQDVTSIPISALHGDNVVTKSDQTPWYEGPSLLSHLEDVYIAGDRNMVDVRFPVQYVIRPHTLEHQDHRSYAGTVASGVMRSGDEVVVLPIGKTTRITAIDGPNGPVAEAFPPMAVSVRLADDIDISRGDMIARTHNQPRITQEFDATVCWMADNAVLEPGRDYVVKHTTRTVRARIAGLDYRLDVNTLHRDKTATALKLNELGRVSLRTQVPLLLDEYTRNASTGSFILIDPDTNGTVAAGMVLRDVSARTPSPNTVRHRSLVTAQDRPPRGKTVWFTGLSGSGKSSVAMLVERKLLEKGISAYVLDGDNLRHGLNADLGFSMADRAENLRRLSHVATLLADCGHLVLVPAISPLAEHRALARKVHADAGIDFFEVFCDTPLQDCERRDPKGLYAKARAGEITHFTGIDSPYQRPKNPDLRLTPDRSIDEQAQEVIDLLESSS(Ag85B nucleic acid sequence) SEQ ID No: 5ATGACAGACGTGAGCCGAAAGATTCGAGCTTGGGGACGCCGATTGATGATCGGCACGGCAGCGGCTGTAGTCCTTCCGGGCCTGGTGGGGCTTGCCGGCGGAGCGGCAACCGCGGGCGCGTTCTCCCGGCCGGGGCTGCCGGTCGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGCGACATCAAGGTTCAGTTCCAGAGCGGTGGGAACAACTCACCTGCGGTTTATCTGCTCGACGGCCTGCGCGCCCAAGACGACTACAACGGCTGGGATATCAACACCCCGGCGTTCGAGTGGTACTACCAGTCGGGACTGTCGATAGTCATGCCGGTCGGCGGGCAGTCCAGCTTCTACAGCGACTGGTACAGCCCGGCCTGCGGTAAGGCTGGCTGCCAGACTTACAAGTGGGAAACCTTCCTGACCAGCGAGCTGCCGCAATGGTTGTCCGCCAACAGGGCCGTGAAGCCCACCGGCAGCGCTGCAATCGGCTTGTCGATGGCCGGCTCGTCGGCAATGATCTTGGCCGCCTACCACCCCCAGCAGTTCATCTACGCCGGCTCGCTGTCGGCCCTGCTGGACCCCTCTCAGGGGATGGGGCCTAGCCTGATCGGCCTCGCGATGGGTGACGCCGGCGGTTACAAGGCCGCAGACATGTGGGGTCCCTCGAGTGACCCGGCATGGGAGCGCAACGACCCTACGCAGCAGATCCCCAAGCTGGTCGCAAACAACACCCGGCTATGGGTTTATTGCGGGAACGGCACCCCGAACGAGTTGGGCGGTGCCAACATACCCGCCGAGTTCTTGGAGAACTTCGTTCGTAGCAGCAACCTGAAGTTCCAGGATGCGTACAACGCCGCGGGCGGGCACAACGCCGTGTTCAACTTCCCGCCCAACGGCACGCACAGCTGGGAGTACTGGGGCGCTCAGCTCAACGCCATGAAGGGTGACCTGCAGAGTTCGTTAGGCGCCGGCTGA (Ag85B amino acid sequence)SEQ ID No: 6MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG (HspX promoter) SEQ ID No: 7CTGCACCGCGCTCTTGATGACATCGGTGGTCACCATGGTGTCCGGCATGATCAACCTCCGCTGTTCGATATCACCCCGATCTTTCTGAACGGCGGTTGGCAGACAACAGGGTCAATGGTCCCCAAGTGGATCACCGACGGGCGCGGACAAATGGCCCGCGCTTCGGGGACTTCTGTCCCTAGCCCTGGCCACGATGGGCTGGTCGGATCAAAGGCATCCGTTTCCATCGATTAGGAGG(Ag85BCysD amino acid sequence) SEQ ID No: 8MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGGFMAITINMVNPTGFIRYEDVEQEAMTSDVTVGPAPGQYQLSHLRLLEAEAIHVIREVAAEFERPVLLFSGGKDSIVMLHLALKAFRPGRLPFPVMHVDTGHNFDEVIATRDELVAAAGVRLVVASVQDDIDAGRVVETIPSRNPIQTVTLLRAIRENQFDAAFGGARRDEEKARAKERVFSFRDEFGQWDPKAQRPELWNLYNGRHHKGEHIRVFPLSNWTEFDIWSYIGAEQVRLPSIYFAHRRKVFQRDGMLLAVHRHMQPRADEPVFEATVRFRTVGDVTCTGCVESSASTVAEVIAETAVARLTERGATRADDRISEAGMEDRKRQGYF (pHspX85BCysD nucleic acid sequence)SEQ ID No: 9CTAGACGCCACCCTCCGGGCCGTTGCTTCGCAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGCCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCATGGGGAGACCCCACACTACCATCGGCGCTAGAGCCGA CTAGAC CTGCACCGCGCTCTTGATGACATCGGTGGTCACCATGGTGTCCGGCATGATCAACCTCCGCTGTTCGATATCACCCCGATCTTTCTGAACGGCGGTTGGCAGACAACAGGGTCAATGGTCCCCAAGTGGATCACCGACGGGCGCGGACAAATGGCCCGCGCTTCGGGGACTTCTGTCCCTAGCCCTGGCCACGATGGGCTGGTCGGATCAAAGGCATCCGTTTCCATCGATTAGGAGGAAGCTTATGACAGACGTGAGCCGAAAGATTCGAGCTTGGGGACGCCGATTGATGATCGGCACGGCAGCGGCTGTAGTCCTTCCGGGCCTGGTGGGGCTTGCCGGCGGAGCGGCAACCGCGGGCGCGTTCTCCCGGCCGGGGCTGCCGGTCGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGCGACATCAAGGTTCAGTTCCAGAGCGGTGGGAACAACTCACCTGCGGTTTATCTGCTCGACGGCCTGCGCGCCCAAGACGACTACAACGGCTGGGATATCAACACCCCGGCGTTCGAGTGGTACTACCAGTCGGGACTGTCGATAGTCATGCCGGTCGGCGGGCAGTCCAGCTTCTACAGCGACTGGTACAGCCCGGCCTGCGGTAAGGCTGGCTGCCAGACTTACAAGTGGGAAACCTTCCTGACCAGCGAGCTGCCGCAATGGTTGTCCGCCAACAGGGCCGTGAAGCCCACCGGCAGCGCTGCAATCGGCTTGTCGATGGCCGGCTCGTCGGCAATGATCTTGGCCGCCTACCACCCCCAGCAGTTCATCTACGCCGGCTCGCTGTCGGCCCTGCTGGACCCCTCTCAGGGGATGGGGCCTAGCCTGATCGGCCTCGCGATGGGTGACGCCGGCGGTTACAAGGCCGCAGACATGTGGGGTCCCTCGAGTGACCCGGCATGGGAGCGCAACGACCCTACGCAGCAGATCCCCAAGCTGGTCGCAAACAACACCCGGCTATGGGTTTATTGCGGGAACGGCACCCCGAACGAGTTGGGCGGTGCCAACATACCCGCCGAGTTCTTGGAGAACTTCGTTCGTAGCAGCAACCTGAAGTTCCAGGATGCGTACAACGCCGCGGGCGGGCACAACGCCGTGTTCAACTTCCCGCCCAACGGCACGCACAGCTGGGAGTACTGGGGCGCTCAGCTCAACGCCATGAAGGGTGACCTGCAGAGTTCGTTAGGCGCCGGCGGATCCATGGCAATAACCATAAATATGGTCAATCCTACCGGATTTATCAGGTATGAGGACGTGGAACAGGAAGCCATGACCAGCGATGTGACGGTGGGCCCCGCACCCGGCCAGTACCAACTGAGCCATCTGCGCTTGCTGGAGGCCGAAGCCATCCACGTCATCCGGGAGGTGGCCGCCGAGTTCGAGCGGCCAGTGCTGTTGTTCTCGGGGGGCAAGGACTCCATCGTCATGCTGCACCTGGCGCTGAAGGCGTTTCGGCCCGGGCGACTGCCGTTCCCGGTCATGCACGTCGACACCGGTCACAACTTCGACGAAGTTATCGCTACCCGAGACGAGTTGGTCGCCGCGGCCGGGGTGCGGCTGGTGGTGGCGTCGGTGCAGGACGATATCGATGCCGGTCGGGTCGTCGAGACCATCCCGTCGCGAAATCCGATACAGACCGTGACGCTGCTGCGGGCCATCCGGGAGAACCAATTCGACGCGGCATTCGGGGGAGCCCGGCGCGACGAGGAGAAGGCCCGCGCCAAGGAGCGGGTGTTCAGCTTCCGCGACGAGTTCGGCCAGTGGGACCCGAAGGCTCAGCGGCCGGAACTGTGGAACCTCTACAACGGACGGCACCACAAGGGCGAGCACATCCGGGTCTTCCCGCTGTCCAACTGGACCGAATTCGACATCTGGTCCTACATCGGCGCCGAGCAGGTCAGGCTGCCGTCCATCTATTTCGCCCACCGGCGCAAGGTGTTTCAGCGCGACGGCATGTTGCTGGCCGTGCACCGGCACATGCAACCGCGAGCCGACGAGCCGGTGTTCGAGGCCACGGTGCGATTCCGCACCGTCGGGGATGTTACCTGCACCGGGTGCGTCGAGTCGTCGGCATCGACGGTCGCGGAAGTCATCGCCGAAACTGCGGTGGCCCGCTTGACGGAGCGCGGGGCGACCAGGGCTGACGACCGGATCTCGGAGGCTGGAATGGAAGACCGCAAGCGGCAGGGATACTTC CAGCTGCACCACCACCACCACCACTGAGTTAACTAGCGTACGATCGACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATGCCATCATGGCCGCGGTGATCAGCTAGCCACCTGACGTCGGGGGGGGGGGAAAGCGACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCCATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAATCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCACTAGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAACGCGTGAGCCCACCAGCTCCGTAAGTTCGGGTGCTGTGTGGCTCGTACCCGCGCATTCAGGCGGCAGGGGGTCTAACGGGTCTAAGGCGGCGTGTACGGCCGCCACAGCGGCTCTTAGCGGCCCGGAAACGTCCTCGAAACGACGCATGTGTTCCTCCTGGTTGGTACAGGTGGTTGGGGGTGCTCGGCTGTCGCTGGTGTTTCATCATCAGGGCTCGACGGGAGAGCGGGGGAGTGTGCAGTTGTGGGGTGGCCCCTCAGCGAAATATCTGACTTGGAGCTCGTGTCGGACCATACACCGGTGATTAATCGTGGTTTATTATCAAGCGTGAGCCACGTCGCCGACGAATTTGAGCAGCTCTGGCTGCCGTACTGGTCCCTGGCAAGCGACGATCTGCTCGAGGGGATCTACCGCCAAAGCCGCGCGTCGGCCCTAGGCCGCCGGTACATCGAGGCGAACCCAACAGCGCTGGCAAACCTGCTGGTCGTGGACGTAGACCATCCAGACGCAGCGCTCCGAGCGCTCAGCGCCCGGGGGTCCCATCCGCTGCCCAACGCGATCGTGGGCAATCGCGCCAACGGCCACGCACACGCAGTGTGGGCACTCAACGCCCCTGTTCCACGCACCGAATACGCGCGGCGTAAGCCGCTCGCATACATGGCGGCGTGCGCCGAAGGCCTTCGGCGCGCCGTCGATGGCGACCGCAGTTACTCAGGCCTCATGACCAAAAACCCCGGCCACATCGCCTGGGAAACGGAATGGCTCCACTCAGATCTCTACACACTCAGCCACATCGAGGCCGAGCTCGGCGCGAACATGCCACCGCCGCGCTGGCGTCAGCAGACCACGTACAAAGCGGCTCCGACGCCGCTAGGGCGGAATTGCGCACTGTTCGATTCCGTCAGGTTGTGGGCCTATCTTCCCGCCCTCATGCGGATCTACCTGCCGACCCGGAACGTGGACGGACTCGGCCGCGCGATCTATGCCGAGTGCCACGCGCGAAACGCCGAATTTCCGTGCAACGACGTGTGTCCCGGACCGCTACCGGACAGCGAGGTCCGCGCCATCGCCAACAGCATTTGGCGTTGGATCACAACCAAGTCGCGCATTTGGGCGGACGGGATCGTGGTCTACGAGGCCACACTCAGTGCGCGCCATGCGGCCATCTCGCGGAAGGGCGCAGCAGCGCGCACGGCGGCGAGCACAGTTGCGCGGCGCGCAAAGTCCGCGTCAGCCATGGAGGCATTGCTATGAGCGACGGCTACAGCGACGGCTACAGCGACGGCTACAACTGGCAGCCGACTGTCCGCAAAAAGCGGCGCGTGACCGCCGCCGAAGGCGCTCGAATCACCGGACTATCCGAACGCCACGTCGTCCGGCTCGTGGCGCAGGAACGCAGCGAGTGGTTCGCCGAGCAGGCTGCACGCCGCGAACGCATCCGCGCCTATCACGACGACGAGGGCCACTCTTGGCCGCAAACGGCCAAACATTTCGGGCTGCATCTGGACACCGTTAAGCGACTCGGCTATCGGGCGAGGAAAGAGCGTGCGGCAGAACAGGAAGCGGCTCAAAAGGCCCACAACGAAGCCGACAATCCACCGCTGTTCTAACGCAATTGGGGAGCGGGTGTCGCGGGGGTTCCGTGGGGGGTTCCGTTGCAACGGGTCGGACAGGTAAAAGTCCTGGTAGACGCTAGTTTTCTGGTTTGGGCCATGCCTGTCTCGTTGCGTGTTTCGTTGCGTCCGTTTTGAATACCAGCCAGACGAGACGGGGTTCTACGAATCTTGGTCGATACCAAGCCATTTCCGCTGAATATCGTGGAGCTCACCGCCAGAATCGGTGGTTGTGGTGATGTACGTGGCGAACTCCGTTGTAGTGCTTGTGGTGGCATCCGTGGCGCGGCCGCGGTACCAGATCTTTAAAT Bold = hspX promoter sequence Bold & underlined = cloning sites Italic = Ag85B-CysD gene Underlined = 6 histidine tag

1.-14. (canceled)
 15. A recombinant or synthetic protein including: afirst region having a sequence encoded by a gene that encodes acomponent of a Mycobacterium sulphate assimilation pathway (SAP); and afurther region having a sequence of a Mycobacterium protein.
 16. Theprotein of claim 15, wherein the first region has a sequence encoded bya CysD gene.
 17. The protein of claim 15, wherein the further region hasa sequence of Ag85B protein.
 18. The protein of claim 15, wherein theprotein has a sequence as shown in SEQ ID No: 2
 19. A nucleic acidencoding a protein of claim
 15. 20. The nucleic acid of claim 19,wherein the nucleic acid has a sequence as shown in SEQ ID No:
 1. 21.The nucleic acid of claim 19, wherein the nucleic acid includes aMycobacterium HspX promoter.
 22. A vaccine or immune stimulatingcomposition for providing an immune response to Mycobacterium in anindividual including: a recombinant or synthetic protein having asequence encoded by a gene that encodes a component of a MycobacteriumSAP; a compound for potentiating an immune response to the recombinantor synthetic protein.
 23. A vaccine or composition of claim 22, whereinthe recombinant or synthetic protein has a sequence encoded by CysDgene.
 24. A vaccine or composition of claim 22, wherein the recombinantor synthetic protein is a protein comprising: a first region having asequence encoded by a gene that encodes a component of a Mycobacteriumsulphate assimilation pathway (SAP); and a further region having asequence of a Mycobacterium protein.
 25. The vaccine or composition ofclaim 22, wherein the compound for potentiating an immune response tothe component of the Mycobacterium SAP includes an adjuvant in the formof MLP and/or DDA.
 26. The vaccine or composition according to claim 22,wherein the recombinant or synthetic protein is provided in the vaccineor composition in the form recombinant bacterial cell having therecombinant or synthetic protein expressed in the cell.
 27. A method forminimising the likelihood of development of a Mycobacterium infection inan individual including: utilising a protein, vaccine or composition ofany one of the proceedings claims to form an immune response to acomponent of a Mycobacterium SAP in an individual; thereby minimisingthe likelihood of development of a Mycobacterium infection in theindividual.
 28. The method of claim 27 wherein the individual does nothave a detectable Mycobacterium infection.
 29. The method of claim 27wherein the individual has one or more symptoms of Mycobacteriuminfection.